Multianalyte molecular analysis using application-specific random particle arrays

ABSTRACT

The present invention provides a method for the generation of novel libraries of encoded magnetic particles from sub-libraries of by the generation of novel sub-libraries of magnetic nanoparticles and encoded particles. The sub-libraries are functionalized on demand are useful in the formation of arrays. The present invention is especially useful for performing multiplexed (parallel) assays for qualitative and/or quantitative analysis of binding interactions of a number of analyte molecules in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationSerial No. PCT/US01/20179, filed Jun. 21, 2001 which claims priority forU.S. Provisional Application Ser. No. 60/213,106 filed Jun. 21, 2000.All the above-referenced applications are expressly incorporated hereinby reference

REFERENCE TO GOVERNMENT GRANT

Some of the inventions described herein were made with governmentsupport under contract No. DAAH01-98-C-R053 awarded by the DefenseAdvanced Research Projects Agency. The government may have certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to the field of materialsscience and analytical chemistry.

The present invention discloses a process for the production oflibraries of encoded magnetic particles, and the formation of planarassemblies inclusive of such particles. The present invention alsodiscloses a platform for the implementation of multistep bioanalyticalassay protocols permitting the integration of sample preparation stepswith the simultaneous analysis of binding interactions between multipletypes of analytes and binding agents.

BACKGROUND OF THE INVENTION

Many bioanalytical procedures such as affinity purification and manybiochemical assays such as immunoassays and DNA hybridization assaysrequire the separation of specific molecules or constituents from acomplex mixture. In the context of molecular and cell biology,magnetizable polymeric particles (“beads”) have been widely used forthis aspect of sample preparation. For example, magnetic beadsdisplaying a short oligo-dT capture probe serve to extract messenger RNA(mRNA) molecules from a cell lysate. Following addition of particles tothe lysate, molecules are captured by hybridization of their poly-Atails to the capture probe, trapped in a magnetic field gradientgenerated by a permanent magnet, retained by the applied magnetic fieldduring the exchange of the lysate for fresh buffer and released intosuspension by removal of the magnetic field (“Biomagnetic Techniques inMolecular Biology,” Technical Handbook, 3^(rd) Edition, DYNAL, 1998). Ina similar manner, magnetic beads displaying antibodies directed againstspecific cell surface antigens serve to selectively extract cells of thedesired type from a given suspension (“Cell Separation and ProteinPurification”, Technical Handbook, 2^(nd) edition, DYNAL, 1997). Arecent example describes a method of magnetic cell separation describesthe use of magnetic particles in conjunction with optical imaging oftrapped cells (A. G. J. Tibbe et al. “Optical tracking and detection ofimmunomagnetically selected and aligned cells” Nature Biotech., 17,1999, 1210-1213).

The integration of assay steps, a principal objective motivating theintroduction of clinical analyzers and other examples of laboratoryautomation, in today's state of the art relies upon a 96-well (orrelated) microwell format of multiple discrete reaction wells toaccommodate standard robotic liquid handling (“pipetting”) and readingof assay signals from individual wells by plate readers. Commercialrobotic pipetting systems have been recently introduced to automatesample preparation based on the use of magnetic beads for separation.However, the integration of sample processing and a highly parallelarray format of analysis by way of microfluidic operations, highlydesirable in connection with the miniaturization of biochemical andanalytical assay procedures, has not been described to date.

The imprinting of multiple binding agents such as antibodies andoligonucleotides on planar substrates in the form of spots or stripesfacilitates the simultaneous monitoring of multiple analytes such asantigens and DNA in parallel (“multiplexed”) binding assays. Theminiaturization of this array format for increasing assay throughput andstudying binding kinetics are described (R. Ekins, F. W. Chu, Clin.Chem. 37, 955-967 (1991); E. M. Southern, U. Maskos, J. K. Elder,Genomics 13, 1008-1017 (1992)). In recent years, this approach hasattracted substantial interest particularly in connection withperforming extensive genetic analysis (G. Ramsay, Nat. Biotechnol. 16,40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21, 33-37 (1999); D.Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent, Nat. Genet. 21,10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. R. Gingeras, D. J.Lockhart, Nat. Genet. 21, 20-24 (1999)).

The principal techniques of array fabrication reported to date include:refinements of the original “spotting” in the form of pin transfer orink jet printing of small aliquots of probe solution onto varioussubstrates (V. G. Cheung, et al., Nat. Genet. 21, 15-19 (1999));sequential electrophoretic deposition of binding agents in individuallyelectrically addressable substrate regions (J. Cheng, et al., Nat.Biotechnol., 541-546 (1998)), and methods facilitating spatiallyresolved in-situ synthesis of oligonucleotides (U. Maskos, E. M.Southern, Nucleic Acids Res. 20, 1679-1684 (1992); S. P. A. Fodor, etal., Science 251, 767-773 (1991)) or copolymerization ofoligonucleotides (A. V. Vasiliskov, et al., BioTechniques 27, 592-606(1999)). These techniques produce spatially encoded arrays in which theposition within the array indicates the chemical identity of anyconstituent probe (BioTechniques 27, 592-606 (1999)). All of thesetechniques of the prior invention require that array formation becompleted prior to initiation of the assay of interest. Therefore, noneof the techniques of array formation of the prior art permit thereal-time formation of arrays subsequent to completion of the bindinginteraction of interest.

Monodisperse magnetic particles confined to planar substrates orinterfaces, and exposed to a uniform magnetic field oriented normal tothe plane of the interface, form a variety of ordered two-dimensionalstructures (W. Wen, L. Zhang and P. Sheng “Planar Magnetic ColloidalCrystals” Phys. Rev. Lett., 85, (25), 5464-5466, 2000; M. Golosovksy, Y.Saado, and D. Davidov “Self-assembly of floating magnetic particles intoordered structures: A promising route for the fabrication of tunablephotonic band gap materials” Appl. Phys. Lett., 75, (26), 4186-4170,(1999); K. Zhan, R. Lenke, and G. Maret “Two-stage melting ofparamagnetic colloidal crystals in two dimensions” Phys. Rev. Letter.,82, (13), 2721-2724, 1999).

Many techniques have been suggested for the synthesis of theseparticles. These techniques attempt to endow the magnetic particles withcertain properties that make them desirable for certain applications.These techniques can be grouped into two categories, the first categoryrelating to synthesis of a magnetic core and the second category relatesto the synthesis of a magnetic shell.

Patents that may be considered of interest in the first categoryinclude:

U.S. Pat. No. 4,358,388 to Daniel et al and U.S. Pat. No. 5,356,713 toCharmot et al. disclose a process which utilizes a suspensionpolymerization approach. One drawback of the process is the difficultyin controlling the mono-dispersity of the resulting magnetic Latex, andthe process does not appear well suited for the generation offluorescent magnetic particles

U.S. Pat. No. 4,654,267 to Ugelstead et al discloses a nitration methodwhich produces particles with a para-magnetic core. Followingmagnetization, the particles are coated with functional polymers toprovide a reactive shell to produce super-paramagnetic particles ofcontrolled morphology, polydispersity, pore size distribution, magneticloading and surface chemistry. The encoding of such particles has notbeen described.

U.S. Pat. No. 4,873,102 to Chang et al discloses a process of formingmagnetic polymer particles containing crystals of magnetite uniformlythroughout the pores. The particles can only be used under hydrophilicconditions.

U.S. Pat. No. 5,356,713 to Charmot et al discloses magnetizablecomposite microspheres which are useful in biological applications butare limited by their size distribution to other applications.

U.S. Pat. No. 5,512,439 to Homes at al discloses monodisperse,super-magnetic particles carrying a plurality of molecules of anoligonucleotide which may be used for single stranded nucleic acids. Theoligonucleotides may be covalently attached or affinity bonded.

U.S. Pat. No. 5,698,271 to Liberti discloses a method for themanufacture of magnetically responsive particles. The particles haveapplications in a variety of preparative and diagnostic techniques.

U.S. Pat. No. 5,866,099 to Owen et al discloses a magnetic-polymerparticle useful in immunoassay techniques and biological/medicalapplications. The particle is produced by co-precipitation of transitionmetals in the presence of a polymer having available coordination sites.

Patents that may be considered of interest in the second categoryinclude:

U.S. Pat. No. 5,736,349 to Sasaki et al discloses a magnetic particlefor an immunoassay method which comprises a core and a coating layer. Anantigen or antibody is bound onto the surface of the coating layer.

U.S. Pat. No. 5,648,124 to Sutor et al discloses a process for theproduction of magnetic particles by hetero-coagulation utilizingoppositely charged core particles and magnetite particles. The dispersedmagnetite may be a coated microparticle which can be further coated withone or more outer polymeric coatings.

U.S. Pat. Nos. 6,013,531, 5,283,079 and 5,091,206 to Wang et al disclosea process for producing magnetically responsive polymer particles. Theparticles comprise a polymeric core particles coated evenly with a layerof polymer containing magnetically responsive metal oxide. The surfaceof these magnetically responsive polymer particles can be coated furtherwith a layer of functionalized polymer. These magnetically responsivepolymer particles can be used for passive o covalent coupling ofbiological material and used as solid phase for various types ofimmunoassays.

Several methods have been described for the synthesis of stainedmagnetic particles. Patents that may be considered of interest includeU.S. Pat. No. 5,395,688 to Wang which discloses a process for producingmagnetically responsive fluorescent polymer particles composed of afluorescent polymer core particle that is evenly coated with a layer ofmagnetically responsive metal oxide. The method utilizes preformedfluorescent polymeric core particles which are mixed with an emulsion ofstyrene and magnetic metal oxide in water and polymerized. A two-stepreactive process such as this suffers from the drawback of possibleinhibition of polymerization by the fluorescent dye or converselybleaching of the fluorescence by the shell polymerization process. Theuse of such magnetic particles containing fluorescent tags for thecalibration of certain solid phase assays has been described in U.S.Pat. No. 6,013,531. Following completion of this step, the particles arecoated with functional polymers to provide a reactive shell.

The creation of core-shell particles from dispersed colloidal matter canbe accomplished by a multistep (layer-by-layer) strategy. The processinvolves step-wise adsorption of charged polymers or nanoparticles andoppositely charged polyelectrolytes onto colloidal particles, exploitingprimarily electrostatic interactions for layer buildup. (Caruso et al“Magnetic Core-Shell Particles: Preparation of Magnetite Multilayers onPolymer Latex Microspheres” Adv. Mater. 1999, 11, 950-953) A shellapplied by electrostratic physisorption is not desirable forbioanalytical assays because it is chemically unstable under changes ofassay conditions, particularly salt concentration, and promotesnon-specific adsorption and denaturation of charged biomolecules; theparticles described in this prior art reference are unsuitable inconnection with the assay formats contemplated herein.

While the foregoing references disclose the use of magnetic particles,none of the prior art particles appear to possess the properties thatare necessary to meet the criteria which are necessary for thesuccessful performance of the assays described herein including apreferred size range, substantial monodispersity, chemicalfunctionalization and synthetic flexibility, the latter permitting therapid construction of libraries of encoded magnetic particles that canbe functionalized on demand, the chemical diversity represented in theselibraries greater than 2. In addition, magnetic particles must meetcertain standards of quality to permit the reproducible assembly ofcustomized arrays to ensure consistent performance in quantitativeassays.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions and processesrelating to the implementation of bioanalytical assay procedures formultiplexed molecular interaction analysis. The processes of theinvention employ a multiplicity of encoded magnetic particles,distinguishable on the basis of their encoding. Such particles displayone or more binding agents which are capable of interacting with one ormore specific analytes upon coming into contact such particles.

The present invention provides a process for the preparation oflibraries of particles which are a composite of an encoded particle anda multitude of magnetic nanoparticles (“composite particles”). Suchcomposite particles can be functionalized to be able to be utilized in aparticular assay. Examples of such functionalization include theinclusion of binding agents such as oligonucleotides, DNA, peptides orproteins. Such composite particles include an optically differentiablecode, such code selected to reflect the nature of the displayed bindingagent, such that the composite particles and their associated bindingagents are distinguishable by real time, in-situ inspection. Custom beadarrays can be fabricated on demand using such libraries. Such arrays areuseful in bioassays, including assays involving multiplexed molecularinteraction analysis.

The bioanalytical assay platform disclosed herein utilizes libraries ofcomposite particles to integrate principal assay steps including samplecapture and preparation, processing and analysis. In a preferredembodiment, analysis is performed in a highly parallel bead array formatin which arrays are assembled in real time following completion ofsample preparation and processing steps.

BRIEF DESCRIPTION OF DRAWINGS

Other objects, features and advantages of the invention discussed in theabove brief explanation will be more clearly understood when takentogether with the following detailed description of an embodiment whichwill be understood as being illustrative only, and the accompanyingdrawings reflecting aspects of that embodiment, in which:

FIG. 1 is an illustration of process flow including the production ofrandom encoded bead arrays and their use in multiplexed assays.

FIG. 2 is an illustration of the functionalization of beads.

FIG. 3 is an illustration of steps in chip design and wafer-scaleproduction.

FIG. 4 is an illustration of on-demand assembly of random encodedarrays.

FIG. 5 is an illustration of palmtop microlab.

FIG. 6 is a schematic illustration of assay and decoding images used inthe random encoded array detection process of the invention.

FIG. 7 is a flow chart summarizing algorithms and steps in the analysisof images.

FIG. 8 is an illustration of steps in the decomposition of assay imagesaccording to bead type by application of the image analysis algorithmsummarized in FIG. 7.

FIG. 9 is an illustration of optically programmable array assembly ofrandom encoded arrays.

FIG. 10 is an illustration of an array composed of random encodedsubarrays.

FIG. 11 is an illustration of stations in an automated chip-scale beadarray manufacturing and quality control process.

FIG. 12 is an illustration of quantitative binding curves for twocytokines.

FIG. 13 is an illustration of array design for polymorphism analysis.

FIG. 14 is a fluorescence micrograph of assay and decoding imagesrecorded from one subarray shown in FIG. 13 in the course ofpolymorphism analysis

FIG. 15 is an illustration of assay results in the form of intensityhistograms obtained from the analysis of assay images such as the oneillustrated in FIG. 14.

FIG. 16 is an illustration of design of a “looped probe” forhybridization assays.

FIGS. 17A and 17B are fluorescence micrographs of assay and decodingimages recorded in the course of the analysis of multiple cytokines.

FIGS. 18A and 18B are illustrations of numerical simulations ofcross-correlations in receptor-ligand systems with multiple competingreceptor-ligand interactions.

FIG. 19 is an illustration of numerical simulations of receptor-ligandassociation and disassociation kinetics.

FIG. 20 is an illustration of integrated sample capture using magneticcapture beads and array-based detection using the random encoded arraydetection process of the invention.

FIG. 21 is an illustration of multi-step assays using compositeparticles to integrate gene-specific capture, on-bead reversetranscription and post-assay array assembly.

FIG. 22 is an illustration of multi-step assay sequence havingapplicability to a wide range of biochemical assays.

FIG. 23(i) is a histogram showing four different types of singleoptically particles and FIG. 23(ii) is a dot plot of eight dual encodedparticles.

FIG. 24 is an illustration of the variation of magnetic loading.

FIG. 25 is an illustration the composite particles of the inventionexposed to a magnetic field.

FIG. 26 is a schematic illustration of the experimental assembly forapplication of a magnetic field.

FIG. 27(i) is a schematic illustration of the 2D structure of the beadassembly before application of a magnetic field. FIG. 27(ii) is aschematic illustration of the 2D structure of the bead assembly afterapplication of a magnetic field of about 100 Gauss. FIG. 27(iii) is aschematic illustration of a close-up of 2D assembly in FIG. 27(ii). FIG.27(iv) is a schematic illustration of the 2D arrangement for a higher(2×) bead concentration under a magnetic field of about 20 Gauss.

FIG. 28 shows results of a streptavidin-biotin binding assay using theencoded and magnetic particles of the invention.

FIG. 29 shows results of a hybridization assay using the encoded andmagnetic particles of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The fabrication of application-specific bead arrays may involve multipleprocesses in a multi-step sequence which may be automated using existingliquid handling technology and laboratory automation. The processdescribed herein and referred to as random encoded array detection(“READ”) includes the fabrication of random encoded arrays as well asthe use of such arrays in bioassays, including assays involvingmultiplexed molecular interaction analysis, including but not limited tothe interaction of analyte and binding agent molecules, as exemplifiedby DNA and protein analysis. Random encoded arrays, as described herein,as well as in U.S. Pat. No. 6,251,691 overcome many of the shortcomingsassociated with processes employing multi-step sequences.

As used herein, the terms “analyte” and “binding agent” refer tomolecules involved in binding interactions. By way of example, analyteand binding agent may include DNA or RNA fragments (e.g.,oligonucleotide), aptamers, peptides, and proteins, antigens and smallorganic molecules. In a particular assay, binding of these fragments totheir complementary sequences (hybridization) is analyzed. In anotherparticular assay, binding interactions between ligands and receptors areanalyzed.

As used herein, the term “particles” refers to colloidal particles andbeads. The term “particle” is also used in connection with the encodedparticles and magnetic particles of the invention.

As used herein, the term “magnetic particle” refers to a particlepossessing a permanent or induced dipole moment.

FIG. 1 provides a schematic overview of the functional components andprocess flow by which custom bead arrays may be prepared and used inperforming multiplexed biomolecular analysis according to the presentinvention. The array is prepared by employing separate batch processesto produce application-specific substrates (e.g., chip at the waferscale), to produce beads that are encoded and functionalized (e.g., atthe scale of ˜10^8 beads/100 μl of suspension) or to produce beads thatare encoded, magnetic and functionalized. The beads subjected torespective quality control (QC) steps prior to array assembly, such asthe determination of morphological and electrical characteristics. Inaddition, actual assays are performed on beads in suspension, beforethey are introduced to the substrate, to optimize assay conditions,generally with the objective to maximize assay sensitivity andspecificity and to minimize bead-to-bead variations. For substrates, QCsteps may include optical inspection, ellipsometry and electricaltransport measurements.

Once the chemically encoded and biologically functionalized beads arecombined with the substrate (e.g., chip), the process described in U.S.Pat. No. 6,251,691 (“LEAPS) in conjunction with the process described inPCT/US97/08159 may be used for rapid assembly of dense arrays on adesignated area on the substrate within the same fluidic phase, avoidingproblems contributing to spot-to-spot as well as chip-to-chipvariability without the need for retooling or process redesign.Furthermore, the bead array format permits chip-independentcharacterization of beads as well as optimization of assay conditions.In addition, multiple bead arrays can be formed simultaneously indiscrete fluid compartments maintained on the same chip. Once formed,these multiple bead arrays may be used for concurrent processing ofmultiple samples. The integration of LEAPS with microfluidics produces aself-contained, miniaturized, optically programmable platform forparallel protein and DNA analysis. U.S. Pat. No. 6,251,691 andPCT/US97/08159 are incorporated herein by reference in their entirety.

In certain embodiments of the present invention, chemical encoding maybe accomplished by staining beads with sets of optically distinguishabletags, such as those containing one or more fluorophore dyes spectrallydistinguishable by excitation wavelength, emission wavelength,excited-state lifetime or emission intensity. The opticallydistinguishable tags made be used to stain beads in specified ratios, asdisclosed, for example, in Fulwyler, U.S. Pat. No. 4,717,655. Stainingmay also be accomplished by swelling of particles in accordance withmethods known to those skilled in the art, (Molday, Dreyer, Rembaum &Yen, J. Mol. Biol 64, 75-88 (1975); L. Bangs, “Uniform Latex Particles,Seragen Diagnostics, 1984]. For example, up to twelve types of beadswere encoded by swelling and bulk staining with two colors, eachindividually in four intensity levels, and mixed in four nominal molarratios. Combinatorial color codes for exterior and interior surfaces isdisclosed in International Application No. PCT/US/98/10719, which isincorporated herein by reference in its entirety.

Beads are functionalized by binding agent molecules attached thereto,the molecule including DNA (oligonucleotides) or RNA fragments, peptidesor proteins, aptamers and small organic molecules in accordanceprocesses known in the art, e.g., with one of several coupling reactionsof the known art (G. T. Hermanson, Bioconjugate Techniques (AcademicPress, 1996); L. Illum, P. D. E. Jones, Methods in Enzymology 112, 67-84(1985). In certain embodiments of the invention, the functionalizedbeads have binding agent molecules (e.g., DNA, RNA or protein)covalently bound to the beads. Beads may be stored in a buffered bulksuspension until needed. Functionalization typically requires one-stepor two-step reactions which may be performed in parallel using standardliquid handling robotics and a 96-well format to covalently attach anyof a number of desirable functionalities to designated beads, asillustrated in FIG. 2. In a preferred embodiment, beads of core-shellarchitecture will be used, the shell composed in the form of a thinpolymeric blocking layer whose preferred composition is selected; andfunctionalization performed in accordance with the targeted assayapplication, as known in the art. Samples may be drawn for automated QCmeasurements. Each batch of beads provides material for hundreds ofthousands of chips so that chip-to-chip variations are minimized.

Substrates (e.g., chips) used in the present invention may be patternedin accordance with the interfacial patterning methods of LEAPS, by forexample, patterned growth of oxide or other dielectric materials tocreate a desired configuration of impedance gradients in the presence ofan applied AC electric field. Patterns may be designed so as to producea desired configuration of AC field-induced fluid flow and correspondingparticle transport. Substrates may be patterned on a wafer scale byinvoking semiconductor processing technology, as illustrated in FIG. 3.In addition, substrates may be compartmentalized by depositing a thinfilm of a UV-patternable, optically transparent polymer to affix to thesubstrate a desired layout of fluidic conduits and compartments toconfine fluid in one or several discrete compartments, therebyaccommodating multiple samples on a given substrate.

In certain embodiments of the invention, the bead array is prepared byproviding a first planar electrode that is substantially parallel to asecond planar electrode (“sandwich” configuration) with the twoelectrodes being separated by a gap and containing an electrolytesolution. The surface or the interior of the second planar electrode ispatterned with the interfacial patterning method. Encoded andfunctionalized beads are introduced into the gap. When an AC voltage isapplied to the gap, the beads form a random encoded array on the secondelectrode (e.g., “chip”). And, also using LEAPS, an array of beads maybe formed on a light-sensitive electrode (“chip”). Preferably, thesandwich configuration described above is also used with a planar lightsensitive electrode and another planar electrode. Once again, the twoelectrodes are separated by the a gap and contain an electrolytesolution. The functionalized and encoded beads are introduced into thegap. Upon application of an AC voltage in combination with a light, thebeads form an array on the light-sensitive electrode.

In certain embodiments, the application-specific bead arrays useful inthe present invention may be produced by picking aliquots of designatedencoded beads from individual reservoirs in accordance with thespecified array composition and “pooled”; aliquots of pooled suspensionare dispensed onto selected substrate (e.g., chips) in a mannerpreventing the initial fusion of aliquots. Aliquots form a multiplicityof planar random subarrays of encoded beads, each subarray representingbeads drawn from a distinct pool and the physical array layout uniquelycorresponding to the identity of aliquots drawn from pooled beadpopulations.

Planar arrays or assemblies of encoded beads on a substrate which arechemically or physically encoded may be used. To this, spatial encodingmay also be added to increase the number of assays that may beconducted. Spatial encoding, for example, can be accomplished within asingle fluid phase in the course of array assembly using LEAPS toassemble planar bead arrays in any desired configuration in response toalternating electric fields and/or in accordance with patterns of lightprojected onto the substrate. LEAPS creates lateral gradients in theimpedance of the interface between silicon chip and solution to modulatethe electrohydrodynamic forces that mediate array assembly. Electricalrequirements are modest: low AC voltages of typically less than 10V_(pp)are applied across a fluid gap of typically 100 μm between two planarelectrodes. This assembly process is rapid and it is opticallyprogrammable: arrays containing thousands of beads are formed withinseconds under electric field. The formation of multiple subarrays, canalso occur in multiple fluid phases maintained on a compartmentalizedchip surface.

The multiplexed assays of the present invention may also be performedusing encoded beads that are assembled, but not in an array, on thesubstrate surface. For example, by spotting bead suspensions intomultiple regions of the substrate and allowing beads to settle undergravity, assemblies of beads can be formed on the substrate. In contrastto the bead arrays formed by LEAPS, these assemblies generally assumelow-density, disorder configurations. However, the combination ofspatial and color encoding attained by spotting mixtures of chemicallyencoded beads into a multiplicity of discrete positions on the substratestill provides a degree of multiplexing that is sufficient for certainbiological assays.

Binding interaction between the binding agent on those beads and ananalyte may be performed either before or after the encoded array isassembled on the substrate. For example, the bead array may be formedafter the assay, subsequent to which an assay image and a decoding imagemay be taken of the array. Alternatively, the beads may be assembled inan array and immobilized by physical or chemical means to produce randomencoded arrays, e.g., with the appearance of the array shown in FIG. 10.The arrays may be immobilized, for example, by application of a DCvoltage to produce random encoded arrays with the appearance of thearray shown in FIG. 10. The DC voltage, set to typically 5-7 V (forbeads in the range of 2-6 μm and for a gap size of 100-150 μm) andapplied for <30 s in “reverse bias” configuration so that an n-dopedsilicon substrate would form the anode, causes the array to becompressed to an extent facilitating contact between adjacent beadswithin the array and simultaneously causes beads to be moved toward theregion of high electric field in immediate proximity of the electrodesurface. Once in sufficiently close proximity, beads are anchored by vander Waals forces mediating physical adsorption. This adsorption processis facilitated by providing on the bead surface a population of“tethers” extending from the bead surface; polylysine and streptavidinhave been used for this purpose.

In certain embodiments, the particle arrays may be immobilized bychemical means, for example, by forming a composite gel-particle film.In one example for forming such gel-composite particle films, asuspension of microparticles is provided which also contains allingredients for subsequent in-situ gel formation, namely monomer,crosslinker and initiator. The particles are assembled into a planarassembly on a substrate by application of LEAPS, e.g., AC voltages of1-20 V_(p-p) in a frequency range from 100's of hertz to severalkilohertz are applied between the electrodes across the fluid gap.Following array assembly, and in the presence of the applied AC voltage,polymerization of the fluid phase is triggered by thermally heating thecell ˜40-45° C. using an IR lamp or photometrically using a mercury lampsource, to effectively entrap the particle array within a gel. Gels maybe composed of a mixture of acrylamide and bisacrylamide of varyingmonomer concentrations from 20% to 5% (acrylamide: bisacrylamide=37.5:1,molar ratio), or any other low viscosity water soluble monomer ormonomer mixture may be used as well. Chemically immobilizedfunctionalized microparticle arrays prepared by this process may be usedfor a variety of bioassays, e.g., ligand receptor binding assays.

In one example, thermal hydrogels are formed using azodiisobutyramidinedihydrochloride as a thermal initiator at a low concentration ensuringthat the overall ionic strength of the polymerization mixture falls inthe range of ˜0.1 mM to 1.0 mM. The initiator used for the UVpolymerization is Irgacure 2959®(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof the silicon substrate. The particle arrays may be formed using suchstructures by, for example, utilizing LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and accumulate particleson the hole arrays. The AC field is then turned off and particles aretrapped into microwells, and are therefor, mechanically confined. Excessbeads are removed leaving behind a geometrically ordered random beadarray on the substrate surface.

When the bead array is immobilized before the assay, the array functionsas a two-dimensional affinity matrix which displays receptors or bindingagents (e.g., oligonucleotides, cDNA, aptamers, antibodies or otherproteins) to capture analytes or ligands (DNA, proteins or other smallcognate ligands) from a solution that is brought in contact with thearray. The bead array platform may be used to perform multiplexedmolecular analysis, such as, e.g., genotyping, gene expressionprofiling, profiling of circulation protein levels and multiplexedkinetic studies.

Substrates (e.g., chips) can be placed in one or more enclosedcompartments, permitting samples and reagents to be transported in andout of the compartments through fluidic interconnection. On-chipimmunoassays for cytokines, e.g., interleukin (IL-6) may be performed inthis format. Serum sample and fluorescent labeled secondary antibodiesare introduced to the reaction chamber sequentially and allowed to reactwith beads immobilized on the chip. FIG. 5 illustrates a design of areaction chamber which may be used in the multiplexed assays accordingto the present invention. Reactions can also be performed in an opencompartment format similar to microtiter plates. Reagents may bepipetted on top of the chip by robotic liquid handling equipment, andmultiple samples may be processed simultaneously. Such a formataccommodates standard sample processing and liquid handling for existingmicrotiter plate format and integrates sample processing and arraydetection.

In certain embodiments, the presence of the analyte-binding agentinteractions are associated with changes in the optical signatures ofbeads involved in the interactions and these optical changes detectedand analyzed. The identities of the binding agents involved in theinteractions are determined by detecting the chemically or physicallydistinguishable characteristic associated with those beads. Preferably,chemically distinguishable characteristics include chemical moleculesincluding flurophore dyes, chromophores and other chemical moleculesthat are used for purposes of detection in binding assays.

The detection of the chemically or physically distinguishablecharacteristic and the detection of optical signature changes associatedwith the binding interactions may be performed while the particles areassembled in a planar array on a substrate, for example, by taking anassay and a decoding image of the array and comparing the two, forexample, a comparison of the assay and the decoding image comprises theuse of optical microscopy apparatus including an imaging detector andcomputerized image capture and analysis apparatus. The decoding imagemay be taken to determine the chemically and/or physicallydistinguishable characteristic that uniquely identifies the bindingagent displayed on the bead surface, for example, by determining theidentity of the binding agents on each particle in the array by thedistinguishable characteristic. The assay image of the array is taken todetect the optical signature of the binding agent and the analytecomplex. In certain embodiments, fluorescent tags (fluorophore dyes) maybe attached to the analytes such that when the analytes are bound to thebeads, the flourescent intensities change, thus providing changes in theoptical signatures of the beads. In certain embodiments, the decodingimage is taken after the beads are assembled in an array and immobilizedand before taking the assay image, preferably before contacting thebinding agents on the beads with an analyte. In certain other examples,the binding interactions occur while the beads are in solution, andassembled into an array afterwards and the decoding and assay images areobtained.

The identity of the binding agent of the binding agent-analyte complexis carried out by comparing the decoding image with the assay image.

In preferred embodiments, images analysis algorithms that are useful inanalyzing the data obtained from the decoding and the assay images.These algorithm may be used to obtain quantitative data for each beadwithin an array. As summarized in FIG. 7, the analysis softwareautomatically locates bead centers using a bright-field image of thearray as a template, groups beads according to type, assignsquantitative intensities to individual beads, rejects “blemishes” suchas those produced by “matrix” materials of irregular shape in serumsamples, analyzes background intensity statistics and evaluates thebackground-corrected mean intensities for all bead types along with thecorresponding variances.

The methods of the present invention may be used for determining theassociation and the dissociation constants e.g., by introducing theanalyte in a time-dependent manner and analyzing the binding as afunction of time, or by washing away the bound analyte in atime-dependent manner and also analyzing the binding as a function oftime.

The methods of the present invention may be used for determining theaffinity constants of analyte-binding agent interactions, fordetermining the number of analyte-binding agent complexes formed

The present invention also provides methods for determining theconcentration of an analyte in a biological sample.

The methods of the present invention may also be used to determiningelements of a co-affinity matrix of a given analyte against a panel ofbinding agents. In one example, the extent of the interaction betweenthe analyte and the binding agents in a panel in competitive,multiconstituent equilibrium reaction may be determined. Determinationof co-affinity constants provides useful applications, as describedbelow.

The successful rate of transplantation for several types of organsdirectly relates to compatibility of Human Leukocyte Antigen (HLA)between donor and recipient. Serological testing of the recipients forthe Panel Reactive Antibodies (PRA) is one of the crucial steps to avoidpossible rejections. Cross-reaction in PRA testing is a very commonphenomenon due to similarity of some HLA antigen structures and thenature of development of these antibodies. In fact, HLA antigens can beorganized into groups based on apparent serological cross-reactivitybetween the groups. These groups are termed Cross-Reactive-Groups(CREGs). In current clinical setting, antibodies from a patient aretested against different antigens in individual reactions. Although areactive pattern of the antibodies can be generated combining theresults from different reactions, the competitive nature of interactionsbetween different antibodies and antigens is not reflected in such apattern. In other cases, several antigens are mixed together for abinding assay. Lack of identification of each antigen in the systemprevents generation of a binding profile. The result is only theaveraged signal from several antigens. In the bead array system, a panelof different antigens is presented to the antibody analytes in acompetitive binding environment, and each antigen can be identifiedthrough its association with different types of beads. Thus, bindingintensity on each antigen in the competitive reactions can be extractedin a single assay. This co-affinity matrix system will provide bindingprofiles for the CREGs and greatly advance the understanding of thenature of the reaction and improve the accuracy for the related clinicaldecisions. For example, a N-antibody and M-antigen system provides amatrix of N×M of possible reactions. It is possible to determine K-nm,the affinity constant governing the interaction between the nth antibodyagainst the mth antigen, where m=1, 2, . . . M, and n=1, 2, . . . N. Forapplications where absolute co-affinity constants are not needed,binding profile will be generated for various antibodies in accordancewith the methods of the present invention and results from a patientsample can be matched to these profiles or combination of theseprofiles.

Co-affinity matrix may also be used to characterize the analyte. Forexample, combination of the coefficients of the co-affinity matrix andknown concentrations of analyte and binding agents participating in theformation of analyte-binding agent complexes serves to define acompetitive binding interaction descriptor, e.g., The molecularinteraction parameter,

${P_{n}\left( R_{m} \right)} = \frac{K_{mn}\left\lbrack L_{n} \right\rbrack}{\sum\limits_{j}{K_{mj}\left\lbrack L_{j} \right\rbrack}}$provides a characterization of the molecular interaction between abinding agent, R_(m), and an analyte, L_(n), in the presence of analytes{L_(j); 1≦j≦N}, all of which exhibit a finite affinity, K_(mj), for thatbinding agent. That is, P_(n), 0≦P_(n)≦1, represents a normalizedspecificity of binding agent R_(m) for analyte L_(n) in amulticonstitutent competitive reaction and serves as a robustcharacterization of that binding agent based on co-affinities displayedin a multiconstituent competitive reaction. See also P. H. von Hippel etal., Proc. Natl. Acad. Sci. USA 83, 1603 (1986), incorporated herein byreference.

The pattern of binding interaction of a analyte against a panel ofbinding agents may be used to characterize the analyte and compare itwith other molecules. In addition, by generating the co-affinity matrixof a analyte using a reference panel of binding agents, such affinitymay be used to determine if a sample later introduced to the panel ofbinding agents contains an impurity by observing the deviation in thebinding pattern.

The present invention also provides use of superparamagnetic particles(“magnetic particles”) as described in U.S. Pat. No. 5,759,820 andEuropean Patent No. 83901406.5 (Sintef), which may then be used inintegrated the sample preparation step with the assay step involvingencoded bead arrays. Both of these references are incorporated herein byreference.

Superparamagnetic particles may be encoded with a chemically orphysically distinguishable characteristic (e.g., fluorescent tag) andused performing bioassays of the present invention. In certainembodiments, the particles are assembled using LEAPS, as withnon-magnetic encoded beads. The encoded nanoparticles can be also beused in array generation, and assayed. The present invention alsoincludes the formation of a planar array of encoded and functionalizedsuperparamagnetic particles on a substrate by application of magneticfield to said particles.

The invention provides a novel process for making color encoded magneticbeads, a simple and flexible one-step process to introduce intopreformed polymeric microparticles a well controlled amount of magneticnanoparticles, prepared in accordance with the procedure describedbelow, along with well controlled quantities of one or more fluorescentdyes. In an embodiment of the present invention, the quantity of themagnetic nanoparticles is controlled to produce magnetic particles thatform an array on a substrate upon application of magnetic field to saidparticles. This process involves swelling the polymer particles in anorganic solvent containing dyes and magnetic nanoparticles and thereforeapplies to any polymer particle which can be subjected to standardswelling procedures such as those disclosed in the prior art offluorescent staining of microparticles Unlike encoding methods in whichthe magnetic material and the fluorescent dyes are each located todifferent areas of the (core/shell) of the magnetic particle, uniformswelling of particles ensures the distribution of magnetic particlesthroughout the interior volume. This process also permits thequantitative control of the nanoparticle as well as dye content over awide range, thereby permitting the tailoring of the particles' magneticsusceptibility as well as fluorescence intensities. An additional methodof the present invention to control the magnetic properties of the hostparticles, other than to control loading, is to tune the size of themagnetic nanoparticles by adjusting the water content of the micellarsynthesis reaction (see below). Physical or chemical coupling ofbiomolecules possible on the particle surface utilizing preexistingfunctional groups. Leaching out of magnetic nanoparticles is readilyeliminated by growing a further polymeric shell on the particle.

The invention also provides a novel process for the production oflibraries of encoded magnetic particles, by a two-step process. Thefirst step in the process involves the production of a sub-library ofmagnetic nanoparticles and the second step in the process involves theproduction of a sub-library of encoded particles. Following sub-libraryformation, as more specifically described below, the members of eachsub-library are endowed with a coupling site and also functionalized

The sub-library of magnetic nanoparticles can be produced by combining ametal oxide dispersion with a monomer solution and forming a polymermatrix of particles encapsulating particles of metal oxide. The metaloxide dispersion may include a metal oxide, used alone or in combinationwith a different metal oxide. The metal oxide may include withoutlimitation, oxides of iron, manganese, cobalt, zinc, nickel and copper,or the like. The monomer solution may include a monomer, used alone orin combination with a different monomer. The monomer may include withoutlimitation, styrene, methyl-methacrylate, acrylamide, ethylene-glycolacrylate, hydroxy-ethyl-methacrylate, vinyl-toluene, divinyl-benzene, orthe like. Alternatively, a polymer solution can be used and may includea polymeric material such as cellulose, protenaceous polymer, glass,agarose, gelatin, or the like. The magnetic nanoparticles may be on anysize and shape, but are preferably, spherical, monodisperse and fromabout 0.01 to about 1 micron, preferably from about 0.05 to about 0.4microns.

The sub-library of encoded particles can be formed by encapsulating anoptical identifier in a polymeric material. The polymeric material mayinclude one polymer, used alone or in combination. The polymers mayinclude without limitation, polystyrene, polymethyl-methacrylate,poly-acrylamide, poly-ethylene-glycol, poly-hydroxy-ethyl-methacrylate,polyvinyl-toluene, poly-divinyl-benzene, brominated polystyrene,polyacrolein, polyethylene, polyurethane, polyvinyl alcohol,polyvinylchloride or combinations thereof, or the like. The opticalidentifier may include one or more dyes with emission wavelengthsbetween 200 nm and 1200 nm. Preferred dyes include but are not limitedto pyro-methane and coumarin dyes. More than one dyes withdistinguishable emission spectra can be used simultaneously and areselected based on their emission spectra.

The encoded particles of the invention may be of any size and shape, butare preferably polymeric, spherical, monodisperse and from about 0.6 toabout 100 microns, preferably from about 1.5 to about 10 microns.

Depending on the application of interest as may be illustrated from theembodiments and examples that follow, each sub-library of the inventioncan be customized. In one embodiment, such customization can beaccomplished by endowing the magnetic nanoparticles with a coupling siteor a functional site which can both be selected based on the applicationof interest. Such sites can be formed by attachment of a molecular groupof interest to the outside surface of the magnetic particle. Thecoupling sites may be formed during the polymerization step and thefunctional sites may be formed at a later stage, prior to formation ofthe composite particles as illustrated below. Molecular entities ofapplicability to the invention for the generation of a coupling siteinclude without limitation any group such as carboxylic, ester, amine,aldehyde, alcohol, or halide, streptavidin, avidin, neutravidin, biotinor the like. Molecular entities of applicability to the invention forthe generation of a functional site include without limitationstreptavidin, avidin, neutravidin, biotin, protein A or the like. Thepre-selection of a molecular entity to endow the magnetic nanoparticlesof the invention with a particular functional site provides the abilityto compile a certain class of sub-libraries which can be useful in theperformance of certain assays. In an illustration of the embodiment ofthis invention, in order to perform an assay for detecting the presenceof a nucleic acid of interest, one would provide the applicablefunctional site to enable the coupling of one or more oligonucleotide orcDNA. In another illustration of the embodiment of this invention, toperform an assay for detecting the presence of a protein of interest,one would provide a functional site to enable the coupling of one ormore antigen or antibody which are specific to the detection ormodulation of the protein of interest. Such functional coupling can beaccomplished prior to or following forming the composite particles ofthe invention.

Generally, the foregoing functional and coupling groups can be attachedto the outside surface of the magnetic nanoparticle by covalentattachment using known methods in the art. Following formation of thesite of interest on the magnetic nanoparticles, the sub-library ofmagnetic nanoparticles can be brought in contact with the sub-library ofencoded particles and the magnetic nanoparticles can be caused to attachto the encoded particles through the coupling site using known method inthe art, while leaving the functional site unattached and available forthe performance of assay of interest.

In another application of the invention, as illustrated in the followingExamples, the metal oxide loading of the composite particles of theinvention can be controlled and adjusted so as to induce a magneticresponse of interest. The foregoing can be accomplished by controllingthe number of magnetic nanoparticles which bind to the surface of theencoded particle.

In another application of the invention, as illustrated in the followingExamples, the optical identifier in the encoded particles can beincorporated in specified ratios to accomplish the objectives of theapplication of interest. The control of the ratio of the opticalidentifier can be accomplished using known methods in the art such asfor example the method disclosed in U.S. Pat. No. 4,717,655.

Following customization of the sub-library of magnetic nanoparticles, asillustrated in the following Examples, a library of composite particlescan be formed by covalently attaching a multitude of functionalizedmagnetic nanoparticle to the outer surface of an encoded particle, theproperties of the particles being pre-selected depending on the assay tobe performed. AS exemplified herein, the chemical diversity representedin the library of the composite particles of the invention is greaterthan 2. In another embodiment, the composite particles of the inventionmay be produced during execution of the assay itself. The assay invokesthe binding interaction between one functional group displayed on amagnetic nanoparticle of the invention and a second molecular groupdisplayed on the encoded particle of the invention to produce thecomposite particle of the invention. In the course of the assay,heterostructures composed of magnetic and non-magnetic particles can beformed as a result of the formation of a complex between functional andmolecular groups following which the composite particles of theinvention can be arranged into array format for detection.

The composite particles of the invention can be manipulated on asubstrate which may be patterned in accordance with the interfacialpatterning methods disclosed in U.S. Pat. No. 6,251,691. Followingpatterning, arrays of the composite particles of the invention can beprepared utilizing the “sandwich” configuration.

The composite particles of the invention can also be manipulated andassembled into ordered arrays in response to magnetic fields. Thepresence of magnetic field gradients induces a force on the compositeparticles (˜½(χV/μo)del (B²)), where χ the magnetic susceptibility of acomposite particle, V its volume, B magnitude of the magnetic field(flux density)) the direction and magnitude of which depends on theexact nature of the magnetic field distribution and the strength of themagnetic field. A proper combination of an uniform axial field andlocalized field gradient generating structures can be used to captureand immobilize (reversibly) the composite particles of the invention inan array format. Such a capability of being able to form 2D arrays aswell as immobilize such particles has particular applicability inconnection with the performance of multi-step assays involving bufferexchange and multiple wash steps. A particularly useful structure forgenerating a uniform axial as well as tunable gradient in the verticaldirection is a pair of electromagnetic coils such that their individualfield directions oppose each other. Hence by adjusting the gap betweenthe coils or the relative position of the sample with respect to thecoils and by varying the individual currents in the coils, the force onthe composite particles of the invention (½(χV/μo) BdB/dz can becontrolled.

As a function of increasing magnetic field strength, ordered planarassemblies of field-dependent number density (or equivalently, averageinter-particle distance) and linear strings of beads oriented normal tothe substrate can be formed.

In a preferred embodiment, planar structures are formed at or near asubstrate by application of a uniform magnetic field oriented normal tothe substrate plane. For typical values of magnetic susceptibility ofcomposite particles with an average diameter of 3.2 μm, prepared asdescribed in Examples provided herein, a field producing a magneticinduction in the range of approximately 1,000 to 2,000 Gauss suffices toproduce planar assemblies. Permanent magnets are available which, whenplaced in immediate proximity to the substrate, generate a field nearthe substrate surface of sufficient field strength to realize thedesired planar configuration of the assembly. Requisite magnetic fieldsconfigurations also can be produced by an electromagnet, for example insolenoid or Helmholtz configuration known to the art; the substrate canbe introduced into the magnet bore or can be placed in immediateproximity to the coil(s) outside of the bore so as to ensure theorientation of the field substantially normal to the substrate plane.The magnetic field generated in such arrangements increases with thecurrent applied to the coils(s) of the electromagnet, thereby providingfor the application of magnetic fields which vary in time, for examplein the form of a linear ramp (for current increasing linearly in time)or sinusoidally (for current varying sinusoidally as a function of time,i.e., an AC current).

Given the field-dependence of the number density within the planarassembly or array, an electromagnet configuration, via real-time controlof the current, thus provides a means to reversibly adjust the numberdensity of the assembly in real time. In a preferred embodiment, an ACcurrent, with frequency in the range 10 Hz to 10 MHz, and more typicallyin the rage of 10 Hz to 10 kHz, is superimposed on a DC current, theamplitude of the AC current chosen to be small compared to the magnitudeof the DC current: the former provides a temporal variation ofinterparticle distance within the assembly while the latter sets theaverage density. This induced density variation of the planar assemblywill set up an electrokinetic response and local flow in the surroundingfluid medium. This magnetic field-induced flow has utility, for exampleby providing local mixing to enhance the kinetics of any reaction ofinterest occurring near the substrate and/or near the particle surface.In addition, spatially modulated magnetic fields can be produced bypatterning the substrate with perm-alloy using methods known to the art.

The invention discloses a platform for the implementation of multistepbioanalytical assay procedures using the libraries of the compositeparticles of the invention to integrate multiplexed molecularinteraction analysis with sample preparation and processing steps.Sample preparation may include capture and separation of desiredanalytes from a given mixture, and sample processing may include anydesired transformations carried out on captured analytes and/or bindingagents. Analysis typically may include the detection and recording ofindicators of the degree of molecular interaction between bead-displayedbinding agents and analyte molecules, an optical signature such asfluorescence or chemiluminescence representing a typical example of suchan indicator; in addition, analysis may include the in-situ decoding theidentities of binding agents associated with individual particles.

In each particular library of the present invention, a binding agent ofinterest in a desired assay is associated with a distinguishable tag inthe form of a color-code. Of specific interest to the present inventionare sub-libraries of encoded particles which enable real-time decoding,and hence the determination of the chemical identity of the bindingagent. In the present invention, pre-selected binding agent areseparately attached to the magnetic nanoparticles of the invention, toproduce a sub-library of functionalized magnetic nanoparticles, and thissub-library is combined with a separately prepared sub-library ofencoded particles serving as bead tags so as to uniquely associate toeach functionalized magnetic nanoparticle contained in the firstsub-library. This process provides unique flexibility in controlling thecomposition and possible permutations of functional groups and tags inthe resulting library.

In one embodiment of the present invention, a library of the compositeparticles of the invention can be utilized in conjunction with standardmicrowell format and liquid handling robotics. The microwells contain aset of particles selected from the library in accordance with therequirements of the assay of interest. Multiplexed interaction analysiscan be performed in one or more wells or other equivalent reactionvessels, preferably under conditions ensuring that the compositeparticles of the invention remain in suspension in order to achievefavorable reaction kinetics and maintain optimal mixing of the reaction.Each of the composite particles present in the reaction is able tointeract with the same mixture of analyte molecules so as to facilitatethe simultaneous formation of multiple binding agent complexes in thismultiplexed assay format in a manner described in the Examples set forthherein. Following completion of the molecular interaction and formationof binding agent complexes on the individual particles, within the setcontained in each reaction vessel, the particles are analyzed anddecoded, for example, by utilizing flow cytometry in a serial mode ofanalysis, to decode the particles and record assay signals one particleat a time, or by transferring one or sets of particles to substratespermitting the formation of planar assemblies for example by permittingthe particles to settle under gravity.

In a preferred embodiment, assay integration is achieved in amicrofluidic environment that eliminates the microwell format. Asdiscussed below, sample capture, subsequent transformations ofbead-displayed binding agents or analytes and real-time formation ofrandom encoded assemblies of encoded particles subsequent to completionof the binding interaction, the latter permitting instant imagingdetection in accordance with the READ process, are all integrated in ahighly parallel assay format. A random encoded bead array, composed ofthe composite particles of the invention formed in real-time byapplication of a magnetic field in accordance with the presentinvention, provides a platform which can be used to perform multi-stepassay sequences.

For purposes of illustration of the invention, four sets of steps(namely capture, transformation, post-transformation array assembly andrandom encoded array detection) can be carried as shown in FIG. 22.These steps are further illustrated in the Examples that follow andprovide the ability to perform an integrated bioanalytical assay. Theassay protocol may be implemented in a microfluidic device of standarddesign, composed of one or more fluidic compartments connected byfluidic conduits and providing for the timed transport of aliquots offluids using standard methods of pumping. In a preferred embodiment,sample capture and transformation are performed in a first compartment;following transformation, beads are transferred to a second compartmentequipped with a magnetic field to complete post-transformation assemblyand the READ process.

Sample of interest and a customized set of the composite particles areintroduced into a first compartment and allowed to interact so as topermit capture of analytes from the sample to bead-displayed bindingagents. Following application of a magnetic trap, one or more washingcycles are performed which involve the replacement of the content of thefirst compartment by aliquots of fresh buffer and finally, to initiatethe transformation step, the introduction of an aliquot of reagentsrequired for the transformation reaction, as described in the Examplesprovided herein.

The following describes in more detail the four steps of the process ofthe invention.

1. Capture

The analysis of biological samples typically begins with complexmixtures such a blood, serum or cell suspensions that contain not onlythe analytes of interest, be they antibodies, antigens, RNA, DNA orother biomolecules, but also a great variety of constituents which mayinterfere with the intended analysis. As with standard analyticalchemical separation, it is generally desirable if not necessary toseparate the analyte fraction of sample from the remainder. Thesuitability of certain magnetic particles for this purpose has beenwidely documented in the prior art, said particles, when used in themicrowell format, generally requiring a high magnetic susceptibility topermit their collection and immobilization within reasonable time inmagnetic field gradients which may be generated in a laboratory settingby use of permanent magnets. In this step, the capture of functionalgroups of interest, including, for example, genomic DNA fragments andthe like to a first set of magnetic particles can be accomplished. Thecapture step is immediately followed by one or more wahing cyclesinvolving the introduction of fresh buffer to replace mobile(non-imobilized) constituents of the original mixture. Inductiveelements also have been described to produce magnetic fields and fieldgradients within a miniaturized environment.

In certain instances, it may not be necessary to distinguish betweendifferent types of analytes of interest; examples include the extractionof total mRNA from cell lysate by hybridization of the common poly-Atail sequence to oligo-dT displayed on standard magnetic particles orthe affinity purification of a class of antibodies, say IgG, by bindingof the Fc portion of the molecule to bead-displayed protein A, as instandard affinity purification.

However, in many instances, it is desirable or necessary to capturemultiple analytes from a given sample in an analyte-specific manner. Inthe absence of a library of the composite particles of the invention,the sample require splitting into multiple aliquots, an additional stepwhich not only has the potential to introduce inaccuracies such aspipetting error or contamination, but require the use of prohibitivelysmall amounts of available sample. In contrast, libraries of thecomposite particles of the invention facilitate multiplexing by way ofsimultaneous capture in an analyte-specific manner. Examples include thesequence-specific capture of multiple mRNA targets of interest from celllysate to matching bead-displayed capture sequences; the selection ofmultiple cDNA targets from a cDNA library; or the capture of multiplerare types of lymphocytes, identified by their respective repertoire ofexpressed cell surface antigens, to bead-displayed monoclonalantibodies. As described in Example 25, sample introduced in thespecific capture step can be produced in a first, non-specific capturestep involving, for example the magnetic nanoparticles of the invention.

In one embodiment of magnetic capture of the composite particles duringwash cycles, permanent magnets known in the prior art may be employed toachieve temporary immobilization. Here, miniaturization of the assayenvironment ensures that particles always reside within a short distanceof typically not more than 100 μm from the nearest bounding surface ofthe reaction vessel, thereby reducing the time required to collectparticles of given magnetic susceptibility from suspension into amagnetic gradient, or, conversely, to minimize the requisite magneticsusceptibility to ensure trapping within a given collection time,typically not more than 5 minutes and preferably not more than 0.5minutes, by a magnetic field and field gradient of given strength. Forthe composite particles of the present invention, the magneticsusceptibility as determined by the constituents of the shell ratherthan the core, and they are therefore particularly well suited tominiaturized assay environments.

One embodiment of the present invention may utilize designs of inductiveelements known to the art for particle trapping at or near planarsubstrate surfaces. In a preferred embodiment, capture is performedwithin a first microfluidic compartment permitting application ofmagnetic fields. For example, a planar fluidic compartment may besandwiched between two permanent magnets which are arranged to provide asufficiently string trap to hold particles against the two boundingsurface of the compartment in the presence lateral flow up to a certainrate, but will release them in the presence of higher flow rates;equivalent magnetic field configurations also are readily produced bycombination of solenoid magnets. A further alternative is provided by anaxially symmetric configuration such as a capillary tube of 10-100 μmdiameter which may be inserted into the center of a quadru-polar fieldto provide trapping.

2. Transformation

The transformation of the captured analytes, or, in some cases relevantto DNA analysis, the analyte-mediated transformation of thebead-displayed probe itself represents an important processing step.Within the context of molecular biology, the most important processingsteps involve several well studied enzyme-mediated modifications of RNAor DNA, notably including, in the former case, reverse transcription,and in the latter case, ligation, extension of bead-displayed probes(also known as primers) or amplification by cycling of extensionreactions, most prevalently by application of polymerase chain reaction(PCR) protocols well known to the art. Specifically of interest to thepresent invention are implementations of on-bead RNA and DNAmodification reactions. As with the capture step, multiplexing ofreactions such as PCR using bead-tagged primers calls for the use oflibraries of the composite particles of the present invention. Anexample is the solid-phase amplification of captured “particle-tagged”fragments. In the transformation step, “particle-tagged” DNA fragmentsare subjected to a set of “multiplex” transformation steps such as forexample PCR amplification. Another example is the polymerase-mediatedextension of bead-displayed probe to produce a bead-displayed cDNAstrand whose composition is complementary to the captured RNA target Ingeneral, it also is possible to create solution-borne products as aresult of the transformation which may then be captured to a second setof the composite particles of the invention as exemplified herein.

Captured protein analytes also may be transformed prior to furtheranalysis. For example, proteins modified to contain a histidinederivative may be modified by chelation to nickel-functionalizedreagents and tags using methods known to the art.

3. Post-Transformation Array Assembly

The present invention utilizes the magnetic field-induced formation ofarrays comprising the composite particles of the invention as a means toproduce random encoded assemblies and arrays of particles within thecontext of a highly bioanalytical parallel assay format in real timefollowing completion of transformation. This process can produce planarassemblies of the composite particles of the invention in designatedregions of the substrate surface to facilitate subsequent imaging ofassemblies, and can produce such assemblies and ordered arrays under thewidely varying conditions ensuring successful molecular interactionanalysis and stability of the resulting binding agent complex, theseconditions pertaining to buffer formulation (salt concentration, pH),presence of additional constituents such as surfactant or adjuvant,temperature and the like. Finite regions of the substrate can bedelineated by interfacial patterning as described in the LEAPS process.In addition, spatially modulated magnetic fields can be produced bysubstrate patterning using, for example, permalloy in accordance withstandard procedures.

The particles of the invention can be assembled into planar assembliesor ordered planar arrays in response to applied magnetic fields. Forexample, particles provided within suspension in a fluidic compartmentbounded by two substantially planar surfaces that are arranged in amutually parallel configuration in a sandwich geometry, when exposed toa magnetic field oriented in a direction normal to the boundingsurfaces, will form a planar assembly. For given particle magneticsusceptibility as controlled by the methods of the invention, orderedplanar assemblies of characteristic number density will form in responseto increasing magnetic field strength; in addition, linear strings ofbeads oriented normal to the substrate (and hence substantially parallelto the applied field) can be formed.

The application of suitably designed magnetic fields generated by aconfiguration of permanent magnets or electromagnets are well known inthe art. Permanent magnets can be designed so as to produce the fieldstrength required to realize the desired configuration of the assembly.Requisite magnetic field configurations can be produced by anelectromagnet, for example in solenoid or Helmholtz configuration knownto the art; the substrate can be introduced into the magnet bore or canbe placed in immediate proximity to the coil(s) outside of the bore soas to ensure the orientation of the field substantially normal to thesubstrate plane.

In another embodiment, a magnetic trap of the type described inconnection with the capture step can be employed to exchange the bufferemployed for molecular interaction analysis for a second buffer that isoptimized to realize conditions ensuring rapid array assembly byapplication of LEAPS.

4. Random Encoded Array Detection

Once assembled, the random encoded array of the composite particles ofthe invention may be imaged to record assay signals and may be decodedto identify binding agents associated with individual beads within thearray by the methods and procedures described herein.

Following formation of an array with the composite particles of theinvention, the array provides a platform which can be used to read theresults of the multi-sep assay sequences. The methods disclosed hereinpermit rapid customization of DNA or protein arrays without the need forprocess redesign and avoid problems contributing to spot-to-spot as wellas chip-to-chip variability. Furthermore, the particle array formatpermits chip-independent characterization of particles as well asoptimization of assay conditions. In addition, multiple particles arrayscan be formed simultaneously in discrete fluid compartments maintainedon the same chip, permitting the concurrent processing of multiplesamples.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes and are notto be construed as limiting this invention in any manner.

EXAMPLES Example 1: Optically Programmable Array Formation

As illustrated in FIG. 9, LEAPS serves to simultaneously assemblemultiple random encoded subarrays and to “drag-and-drop” these subarraysinto separate, but proximate locations on the chip within a common,enclosed liquid environment. Two sets of beads (2.8 μm Oligo-(dT)₂₅,Dynal, Oslo, Norway), dispensed from separate reservoirs A and B, weresimultaneously assembled into distinct subarrays within the same fluid;sub-arrays were then simultaneously placed into desired destinations asdirected by spatially varying illumination profiles which were generatedand projected onto the substrate by a PC-programmable illuminationpattern generator (described in U.S. Ser. No. 09/397,793, filed Sep. 17,1999, which is incorporated herein by reference in its entirety). Thisdrag-and-drop operation reduced the separation between the twosub-arrays from approximately 250 μm to 20 μm. Beads were moved at 5V_(pp) at a frequency of 2 kHz; total power projected onto the substratesurface was ˜5 mW. The combination of chemical and spatial encodingpermits a given set of chemical bead markers to be used multiple timesand reduces the demands placed on either encoding dimension whilefacilitating the realization of large coding capacities.

Example 2: Array Formation on Patterned Surface

Illustrated in FIG. 10 is an array of encoded beads assembled on apatterned silicon chip using an AC voltage of 1-2 V_(pp) and a frequencyof 100-150 Hz, applied across a 100 μm electrode gap filled with anaqueous bead suspension; a thermal oxide (˜1000 Å) on the substrate waspatterned by etching the oxide to a thickness of 50-100 Å in a set ofsquare features (˜30×30 μm²) on 130 μm centers; arrays of similar layoutalso can be produced in response to suitable illumination patterns. Eachsub-array shown here contains approximately 80 beads coupled withanti-cytokine monoclonal antibodies. Carboxylate-modified polystyrenebeads of 5.5 μm diameter (Bangs Laboratory, Fishers, Ind.) were stainedwith a combination of two types of fluorescent dyes and were thenfunctionalized with anti-cytokine-mAb. The assembly process ensurescollection of all beads at the substrate surface. Bead encoding was asfollows: IL-2 (Bright Red); IL-4 (Dim Red); IL-6 (Bright Green); M-CSF(Dim Green) and TNF-α (Yellow).

Example 3: Formation of Arrays of Magnetic Nanoparticles

Colloidal particles exhibiting a finite diamagnetic susceptibility, whendisposed on a planar substrate can be assembled into ordered arrays inresponse to increasing magnetic fields. Commercially availablesuperparamagnetic particles (Dynal, Oslo, NO), dispersed from a fluidsuspension onto the planar surface of the lower of two parallel boundingsurfaces of a fluid cell (“sandwich” geometry), when exposed to ahomogeneous axial magnetic field (oriented normal to the substrateplane), will form ordered assemblies. As a function of increasingmagnetic field strength, and for given diamagnetic susceptibility of theparticles as controlled by the manufacturing process known to the art,ordered planar assemblies and linear strings of beads oriented normal tothe substrate can be formed. Permanent magnets can be designed so as toproduce the field strength required to realize the desired configurationof the assembly. Requisite magnetic field configurations can be producedby an electromagnet in solenoid or Helmholtz configuration known to theart; the substrate can be introduced into the magnet bore or can beplaced in immediate proximity to the coil(s) outside of the bore so asto ensure the orientation of the field substantially normal to thesubstrate plane. Spatially modulated magnetic fields can be produced bypatterning the substrate with permalloy using methods known to the art.

Example 4: Formation of Random Bead Assemblies

Aliquots of solution containing suspended beads were placed onto severaldistinct positions on a planer substrate of silicon capped with a thinsilicon oxide layer (other substrates may be used here). Beads wereallowed to settle under gravity to form random assemblies. To delineatediscrete positions on the substrate, one of the following two methodswere used. According to the first method, a silicon gasket (of 250 μmthickness), displaying a grid of multiple round holes of 1 mm or 2 mmdiameter (Grace Bio-labs, Bend, Oreg.) is placed on the hydrophilicsurface to define microwells (of 0.25 to 0.5 ul volume) for multiplediscrete samples of bead suspension. According to the second method,small aliquots of fluid containing beads (0.2 ul to 0.5 ul in volume)are directly placed onto a hydrophilic surface in one or more designatedareas so as to ensure formation of discrete droplets; spacers are notneeded in this case. As solvent evaporates (at room temperature or, forrapid drying, at elevated temperature (about 60 C), beads are left inrandom positions on the substrate. DNA polymorphism reactions have beentested in assemblies formed in both manners. Optionally, beads settlingunder gravity may be immobilized by chemical capture layers provided onthe substrate. An application of random bead assemblies to determineaffinity constants in a multiplexed format is described in Example 6.

Example 5: An Automated Chip-Scale Array Manufacturing Rocess

As illustrated in FIG. 11, the process involves liquid handling andpipetting of beads onto chips mounted in single-chip cartridges ormulti-chip cartridges. Bead arrays are formed using methods such asthose in Examples 1, 2 or 3., followed by array immobilization anddecoding. The resulting decoding images are stored for later use alongwith an optional chip ID (“bar code”).

Example 6: Determination of Affinity Constants by Post-Assay Analysis ofBead Assemblies

Quantitative binding curves for the cytokines TNF-α and IL-6. Bindingcurves were generated by performing sandwich immunoassays usingchemically encoded beads in suspension, said suspensions being confinedto one or more reaction compartments delineated on-chip, or in one ormore reaction compartments off chip. By completing the reaction withbeads maintained in suspension, assay kinetics similar to homogeneousassays can be attained. Following completion of the binding reaction,beads were assembled on chip to permit multiplexed quantitative imageanalysis. Random assemblies prepared according to Example 4 or orderedbead arrays prepared according to Example 1 or 2 may be used. Anadvantage of ordered, dense assemblies produced by the methods ofExamples 1 or 2 is the higher spatial density and higher assaythroughput attained by processing a greater number of beads.

As an illustration, FIG. 12 displays quantitative binding curves forTNF-α and IL-6, obtained from randomly dispersed beads. Acommercial-grade 8-bit video-CCD camera (Cohu, San Diego, Calif.) wasused in a mode permitting multi-frame integration. The range ofconcentrations of antigen used in the two assays was 700 fM to 50 pM forTNF-α and 2 pM to 50 pM for IL-6. At each concentration, the number ofmolecules bound per bead was estimated by comparison with calibrationbeads coated with known quantities of Cy5.5-labeled BSA per bead;requisite adjustments were made to account for differences influorescence quantum efficiency between labeled secondary antibodies andBSA.

This format of analysis permits the determination of the affinityconstant, K_(A)=[LR]/([R₀-LR][L]), where, in accordance with the law ofmass action, [LR] denotes the number of receptor-ligand pairs per beadand [L] denotes the solution concentration of ligand. By specifying thenumber of beads per ml, n_(B), and specifying a value for [R₀] in termsof the number of receptors per bead, theoretical binding curves,computed for given K_(A), are compared to a plot of the number of boundmolecules per bead as a function of bulk ligand concentration. Theabsolute number of ligands bound per bead may be determined for givenbulk concentration by measuring the mean fluorescence intensity per beadand referencing this to the fluorescence intensity recorded fromcalibration beads included in the array.

The estimated number of molecules bound per bead is compared totheoretical binding curves derived from the law of mass action. Thethree curves shown correspond to values of the affinity constant, K_(A),of 10¹¹/molar, 10¹⁰/molar and 10⁹/molar, respectively. The initialnumber of antibodies per bead, R₀, equals 2×10⁵/bead and n_(B)=10⁵/ml.Each data point represents the average of three replicates, with anassay-to-assay variation of <45%. Setting the assay sensitivity tocorrespond to that level of fluorescence which yields a signal-to-noiselevel of unity in the assay images, the sensitivity of the cytokineassays characterized in FIG. 12 is set at ˜2,000 bound ligands/bead,corresponding to respective detected concentrations of 700 fM for TNF-αand 2 pM for IL-6.

While commercial ELISA kits use enzymatic amplification to enhancesensitivity, at the expense of additional complexity relating to assayconditions and controls, our bead array assay format, even withoutenzymatic amplification, our on-chip assay format permits monitoring ofcytokines at circulating levels (Normal TNF-α level in serum is 50-280fM and normal IL-6 level in serum is 0-750 fM.

www.apbiotech.com/technical/technical_index.html), providing a dynamicrange which approaches that of standard, i.e. amplified single-analyteELISA assays (Assay kits of R & D Systems and Amersham (not the recentHigh-Sensitivity assays). Further improvements at hardware and softwarelevels are possible.

Example 7: Genotyping by Polymorphism Analysis

To illustrate the application of the present invention to theimplementation of genotyping, FIG. 13 shows the design of the assay inwhich five pairs of 20-mer binding agents corresponding to fourpolymorphic regions of a gene were coupled to color-encoded beads. Thepairs of binding agents α1, α2 and β1, β2 each display a singlenucleotide difference in their respective sequences; the pair δ3, δ4displays a difference of three nucleotides, the binding agents in theset γ1, γ3, γ3, γ4 display small insertions and deletions. The tenbinding agents were are divided into two subgroups of five which wereincorporated into two subarrays. In this example, there are severalhundred beads for each type. Following bead immobilization, an on-chiphybridization reaction was performed in TMAC buffer (2.25 Mtetramethylammonium chloride, 37 mM Tris pH 8.0, 3 mM EDTA pH 8.0, and0.15% SDS) at 55° C. for 30 min. The analyte is a 254-base PCR fragmentproduced from a patient sample and fluorescently labeled at the 5′-primeend with BODIPY 630/650 (Molecular Probes, Eugene, Oreg.). Imageacquisition was performed after replacing the assay buffer with freshTMAC buffer.

FIG. 14 shows decoding and assay images for one subarray. Each beadshown in the assay image obtained after hybridization is analyzed todetermine fluorescence intensity and bead type; as with the cytokineassay, the latter operation compares assay and decoding images using atemplate matching algorithm. FIG. 15 displays the resulting intensityhistograms for each bead type: in these histogram plots, the horizontalaxis refers to relative signal intensity from 0 to 1 and the verticalaxises refer to bead numbers. The histograms show that most of the beadsdisplaying probe α1 bind no analyte while most of the beads displayingprobe α2 exhibit significant binding; the mean signal level of α2-beadsexceeds that of α2-beads by a factor of ˜3.2, indicating that analytecontains DNA sequences complementary to α2 but not α1. For the patientsample presented here, the histogram indicates a genotype of the analyteDNA characterized by complementarity to binding agents α2, β2, γ3, γ4and δ4 in the polymorphic region of the gene.

Example 8: Gene Expression Analysis: cDNA Fragments

The method of the present invention has been used to fabricate arrayscomposed of beads displaying oligonucleotides as well as DNA fragments(e.g., up to ˜1,000 bases in length). Strands were biotinylated atmultiple positions by nick-translation and were attached tostreptavidin-functionalized beads (M-280, Dynal, Oslo, NO). Arrays wereformed using an AC voltage of 800 Hz at 10V_(pp).

Example 9: Looped Probe Design for Universal Labeling

A looped probe design in FIG. 16 takes advantage of fluorescence energytransfer to obviate the need for labeled target. As with the molecularbeacon design (S. Tyagi, D. P. Bratu. F. R. Kramer, Nature Biotech. 16,49-53 (1998)), the probe in FIG. 16 assumes two different states offluorescence in the closed loop and open loop configurations, but incontrast to the molecular beacon contains a portion of its binding motifwithin the stem structure to permit molecular control of stringency incompetitive hybridization assays.

Example 10: Quantitative Multiplexed Profiling of Cytokine Expression

FIG. 17 displays a pair of assay and decoding images recorded from asingle random array in a multiplexed sandwich immunoassay. An arraycontaining five distinct types of beads, each displaying a monoclonalanti-cytokine antibody (mAb), was exposed to a sample solution (such asserum) containing two cytokine antigens (Ag). Subsequent addition ofCy5.5-labeled secondary antibodies (pAb*) results in the formation ofternary complexes, mAb-Ag-pAb*. The on-chip immunoassay was performed byadding 300 μl of sample with 7 nM cytokines in assay buffer (20 mM NaPipH 7.4, 150 mM NaCl, 10 mg/ml BSA) to the bead array immobilized on thechip, and allowing the reaction to proceed at 37° C. for one hour. Thebuffer was replaced by adding 12 nM solution of labeled secondaryantibodies in assay buffer. After one hour of incubation at 37° C.,fresh buffer was added on top of the chip and image acquisition wasperformed. Antibodies and antigens used in the assays were obtained fromR&D Systems (Minneapolis, Minn.); the secondary antibody was labeledwith Cy5.5 using a standard kit (Amersham Pharmacia Biotech, Piscataway,N.J.).

The decoding image FIG. 17B shows five types of beads in a false-colordisplay with the same encoding pattern as that of FIG. 10. All beads areof the same size (5.5 μm diameter); the apparent difference in the sizeof beads of different types in the decoding image is an artifactreflecting different internal bead staining levels and “blooming” duringCCD recording of the decoding image. Comparison (using the imageanalysis methods disclosed herein) of the decoding image with the assayimage in FIG. 14A reveals that active beads, of yellow and bright greentypes, captured TNF-α and IL-6, respectively. This assay protocol hasbeen extended to the following set of twelve cytokines: IL-1α, IL-1β,IL-2, IL-4, IL-6, TGF-β1, IL-12, EGF, GM-CSF, M-CSF, MCP-1 and TNF-α.The on-chip immunoassay requires no additional washing other thanchanging reagent solutions between assay steps. Comparison between assayand decoding images shows that two different cytokines were present inthe sample, namely IL-6 and TNF-α The pre-formed arrays described inthis example also permit the determination of affinity constants in amanner analogous to the analysis described in Example 6.

Example 11: Aptamers for Protein Profiling

Aptamers may be selected from large combinatorial libraries for theirhigh binding affinities to serum proteins (L. Gold, B. Polisky, O.Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 64: 763-797. (1995)). Randomencoded arrays of aptamer-functionalized beads would serve to monitorlevels of serum proteins; correlations in binding patterns on the array(see also Example 10) may serve as a phenotype of disease states.

Example 12: Mixed DNA—Protein Arrays

Of significant interest to genomic functional analysis is the fact thatthe method of the present invention accommodates protein and DNA arrayswithout change in array manufacturing methodology. Specifically, mixedarrays composed of beads displaying DNA and corresponding proteins canbe used to analyze the gene and gene product within the same fluidsample.

This has been demonstrated for a combination of immunoassay and DNAhybridization. For example, a mixed array composed of beadsfunctionalized with anti-cytokine monoclonal antibodies (mAb) and witholigonucleotides was produced. Two sequential assays were performed onthis single chip. First, an immunoassay was performed in accordance withthe protocol described in Example 10. Following completion of theon-chip immunoassay, image is acquired and the DNA analyte was added tothe hybridization buffer (2×SSC, 1×Denhardt's) at a final concentrationof 20 nM and allowed to react at 37° C. for 1 hr. Fresh hybridizationbuffer was added to the chip and image acquisition was performed torecord of the additional hybridization assay.

Example 13: Affinity Fingerprinting

The analysis of receptor-ligand interactions relevant to prior artmethods assumes ideal specificity. That is, only the ideal situation isconsidered of a single ligand present in solution reacting with itsmatching receptor and vice versa. However, in most multiple assaysystems, a considerable level of cross-reactivity may exist. That is,any single ligand may associate with several receptors, while any singletype of receptor may have a finite affinity towards more than oneligand.

The present invention includes a model that is developed to analyzemultiplexed READ assays for such a system under the followingassumptions: each of these reversible reactions is characterized by itsown affinity constant; no reaction occurs between the bulk species;there is no interaction between the complexes formed on the surface.These assumptions can be relaxed, at the expense of increasing thecomplexity of modeling, by accounting for reactions in the bulk andbetween the surface species. The standard reaction-diffusion equationfor single receptor-ligand pair formation [R. W. Glaser, Anal. Biochem.213, 152-161 (1993)], is generalized to allow for multiple reactions ateach bead surface:

$\begin{matrix}{{\frac{\partial\left\lbrack {L_{i} \cdot R_{j}} \right\rbrack}{\partial t} = {{{k_{{on},{ij}}\left\lbrack L_{i} \right\rbrack}\left( {\left\lbrack R_{j,0} \right\rbrack - {\sum\limits_{n,m}\left\lbrack {L_{m} \cdot R_{n}} \right\rbrack}} \right)} - {k_{{off},{ij}}\left\lbrack {L_{i} \cdot R_{j}} \right\rbrack}}}{{\forall i},j,{L_{i} \equiv {L_{i}\left( {t,x,0} \right)}}}} & (2)\end{matrix}$

The first term on the right of Eq. (1) describes the association ofligands and receptors into complexes and involves of concentration offree sites on the surface. The second term describes the disassociationof complexes by way of release of ligands, thereby freeing up receptorsites for further reaction. Since a maximum of (i×j) bimolecularcomplexes can form, there could be as many boundary conditions generatedfrom the above equation. For the equilibrium case, the left hand-side ofEq. (1) is set to zero, and the matrix of coaffinities,[K_(ij)]=k_(on,ij)/k_(off,ij), can then be defined to accommodatecross-reactivities between multiple species in the bulk and on thesurface. In a batch reactor under equilibrium conditions, we may solvethe system of differential equations to obtain the number of moleculesof each ligand bound on beads of each type.

L₁ Ligand concentration  10 pM L₂ Ligand concentration 100 pM R₀₁Initial receptor concentration 1 × 10⁴/bead R₀₂ Initial receptorconcentration 1 × 10⁴/bead n_(B1) Bead number density 1 × 10⁴/ml n_(B2)Bead number density 1 × 10⁴/ml [K] Coaffinity matrix [1 × 10¹¹ 1 × 10⁹ 1× 10⁸ 1 × 10¹¹] 1/mole

As an illustrative example, the ligand distribution has been calculated(from the model in Eq (1)) for a reference set of two ligands and twotypes of receptors immobilized on two different sets of beads. Thecoaffinity matrix is assumed known for each ligand-receptor combinationin the reference set; to investigate the detection of a third ligand, itis assumed here that diagonal elements of the 2×2 matrix, [K_(ij)], arelarge compared to off-diagonal elements. The presence of a third ligandin the reactor alongside the two original ligands perturbs theequilibria between the various complexes and the reactants in thereference system, and for ligand molecules tagged with fluorescentlabels, the intensity observed from the perturbed system differs fromthat observed in the reference case.

FIG. 18A shows the reference case in which the concentrations andcoaffinity matrix were set to the values shown in the accompanyingtable; the bead intensity was defined on a linear scale of 0-255, thelatter representing the intensity of the brightest beads. FIG. 18A showsthe contribution of each ligand to the bead intensity. Due to the lowerconcentration of L₁, the intensity of Bead 1 is less compared to Bead 2,cross-reactivities are essentially undetectable.

Next, the system was perturbed with a third ligand, taking theconcentration L₃ to be 1 pM and assuming that the new ligand hasconsiderable amount of cross-reactivity with each of the receptors;K_(3,1)=1×10¹¹/M, K_(3,2)=1×10¹⁰/M. Calculation of the fluorescentintensity of each bead in the presence of the third ligand yields thepattern in FIG. 18A which reveals an increase in the intensity of Bead 1due to the third ligand, while leaving the intensity of Bead 2unaffected due to the higher concentration of L₂ in the system and thelower affinity of L₃ to R₂. Thus, L₃ may be detected under the conditionthat it has a relatively high affinity to one of the receptors and is insignificant amount compared to the competing ligand.

The evaluation of the coaffinity matrix (and comparison with theoreticalmodeling as disclosed herein) under conditions in which a mixture ofligands is permitted to interact with a multiplicity of receptorsarranged in a random encoded bead array format provides a methodology toestablish a characteristic feature set of cross-correlations in themutual competitive binding affinities of multiple ligands and receptors.These co-affinities provide a robust means to characterizereceptor-ligand binding equilibria by their affinity fingerprintingpatterns. Deviations from well-defined reference cases also permitdetection of “perturbing” ligands in solutions.

Example 14: Multiplexed Analysis of Reaction Kinetics

As illustrated in the foregoing examples, extensive washing generally isnot required to discriminate beads from a background of solutionfluorescence. Consequently, assay image sequences may be recorded in ahomogeneous assay format to document the evolution of a binding reactionand to determine kinetic data for each of the binding reactionsoccurring.

Homogeneous binding assays may be performed in simple “sandwich” fluidiccartridges permitting optical microscopic imaging of the bead array andpermitting the introduction of an analyte solution into a chambercontaining a random encoded array of beads. More generally, the arrayalso may be exposed to an analyte or other reaction mixtures underconditions of controlled injection of fluid aliquots or continuous flowof reactants or buffer. Using theoretical modeling, optimal combinationsof relevant performance control parameters of this bead array reactormay be identified to minimize the time to equilibration or to maximizethe portion of analyte captured by the array [K. Podual and M. Seul, TMKP-99/02]. Flow rate can be controlled by any of a number of availablepumping mechanisms [M. Freemantle, C&EN, 77: 27-36].

TABLE List of parameters used in simulations (FIG. 18) Parameter, unitsValue Initial Receptor Coverage c_(R, 0), moles/m² 8 × 10⁻⁹ Vol FlowRate, Q, μl/s 1.0 Diffusivity, D, cm²/s 1 × 10⁻⁷ ON-Rate, k_(on), /(M s)1 × 10⁵  Affinity Constant, K_(A), /M 1 × 10¹¹ “Sandwich” Reactor GapSize H, mm 0.1 Reactor Length, L, mm 10 Reactor Width, W, mm 10

The analysis of image sequences permits kinetic data to be generatedfrom which ON-rates and OFF-rates are determined with the aid of atheoretical model of the reaction-diffusion kinetics of the typeillustrated in the foregoing example in FIG. 19. FIG. 19A displaysstages in an adsorption-desorption cycle involving solution-borneanalytes and a bead array immobilized at the bottom of a “sandwich”reaction chamber. The first panel depicts the initiation of theadsorption process; the second panel depicts the state of the reactorclose to equilibrium when most of the beads have reached equilibrium;the last panel depicts the state of the reactor under the desorptioncycle in which ligand-free fluid is injected and adsorbed moleculesdesorb from the bead surface. FIG. 19B displays theadsorption-desorption kinetics of a single receptor-single ligand systemobtained by numerical solution of a reaction-diffusion system for asingle type of receptor-ligand reaction; two cases of differentconcentrations of ligand are shown. Parameters used in the simulationare listed in the accompanying Table.

In contrast to prior art methods [D. G. Myszka, Curr. Opin. Biotechnol.8: 50-57.], the present method relies on imaging and permitsmultiplexing. In addition, generalized models of the type introduced inExample 6 permit the analysis of complex binding kinetics for multiplesimultaneous receptor-ligand interactions even in the presence ofcross-reactions between multiple ligands and receptors.

The ability to monitor reaction kinetics in an array format will enableseveral approaches to enhancing the specificity of receptor-ligand orbinding agent-analyte interactions in complex mixtures. For example,temperature programming may be invoked to enhance the specificity of DNAhybridization reactions. Similarly, the stringency of conditions appliedto a hybridization reaction may be varied while the array response isbeing monitored; for example, hybridization may be conducted in ahybridization buffer under conditions leading to excess “non-specific”binding; specificity is enhanced by switching to a wash buffer ofincreasing stringency while monitoring the array response.

Example 15: Multi-Step Assay Sequences Using Encoded Arrays of MagneticParticles

Methods and apparatus using biochemically functionalizedsuper-paramagnetic particles for sample preparation in molecular andcellular biology and for a variety of enzyme-catalyzed on-bead reactionshave been described [“Biomagnetic Techniques in Molecular Biology”,Technical Handbook, 3^(rd) Edition, 1998, Dynal, Oslo, NO]. Thesebead-based methods can be combined with the Random Encoded ArrayDetection format of the present invention to implement multi-stepon-chip assay manipulations.

For example, FIG. 20 illustrates the integration of a sequence of stepsin a miniaturized format for multiplexed genotyping using a single chipwith multiple compartments. First, cells are captured from a patientsample by affinity selection using functionalized magnetic beads, cellsare lysed electrically or chemically in a first compartment, and genomicDNA is captured to the surface of a multiplicity of magnetic beads bynon-specific binding; next, beads are collected by magnetic force into asecond compartment which is in fluidic contact with the firstcompartment, within which the beads and DNA are washed with desiredbuffers; next, beads are further transferred to a location where PCR isperformed using bead-coupled DNA as a template; multiple PCR strategiesknown in the art are available for this step (F. Fellmann, et. al.,Biotechniques, 21:766-770); next, PCR products released into arecaptured by hybridization to a pre-assembled random encoded arraydisplaying binding agents that are specific to different polymorphismtargeted by the PCR amplification.

The use of encoded magnetic particles in conjunction with the opticalprogrammability of LEAPS confers the ability to form reversiblyimmobilized arrays and to conduct programmable multi-step assaysequences under conditions in which beads are used in suspension whenthis is most favorable, for example to enhance reaction kinetics, andarrays are formed in real-time when this is most favorable, for exampleto provide a highly parallel format for imaging detection and assayread-out.

For example, as illustrated in FIG. 21, the following sequence of stepscould be integrated in a miniaturized format for the formation of a cDNAbead array. First, a pool of encoded magnetic beads, each bead typedisplaying a gene-specific probe, is introduced to an mRNA pool, andmRNA molecules are hybridized to their corresponding beads; next,on-bead reverse transcription (RT) is performed using bead-attached mRNAas template [E. Horenes, L. Korsnes, US 005759820]; next mRNA isreleased from the beads; next beads are directed to the surface of acustom-designed chip and a cDNA bead array is formed using LEAPS. Suchan array could serve to display binding agents in a gene profilingexperiment using another set of mRNA as the target. Alternatively, thecDNA array could be analyzed for its own expression by applying a poolof labeled DNA binding agents to profile the genes of interest withinthe array.

Example 16: Synthesis of Super-Paramagnetic Iron Oxide γ-Fe₂O₃(Maghemite) Particles

The synthesis was carried out in reversed micellar solutions composed ofthe anionic surfactant, bis(2-ethylhexyl)sodium sulfosuccinate (AOT) andisooctane (Kommareddi et al., Chem. Mater. 1996, 8, 801-809) obtainedfrom Aldrich Chemical Co., Milwaukee, Wis. Stock solutions of 0.5M AOTwere used in preparing the reversed micellar solutions containing thereactants FeSO₄ (Sigma Chemical Co., St. Louis, Mo.) and NH₄OH (SigmaChemical Co., St. Louis, Mo.). Specifically, 0.45 ml of 0.9M FeSO₄ wasadded to 5 ml of 0.5M AOT in isooctane, separately 0.45 ml of NH₄OH wasadded to 5 ml of 0.5M AOT in isooctane. The reaction was initiated byadding the NH₄OH reversed micellar solution to the FeSO₄ reversedmicellar solution under vigorous stirring. The reaction was allowed toproceed for 2-3 hrs and then the solvent was evaporated at ˜40° C. toobtain a dry surfactant iron oxide composite. This composite wasre-dispersed in the organic solvent of choice to give a deep red coloredtransparent solution.

Example 16a: Synthesis of Magnetic Nanoparticles with a Functional Site

This Example illustrates the creation of a functional site on a magneticnanoparticle. A surfactant was dissolved in oil to obtain a reversemicellar solution which was used to synthesize the magneticnanoparticles of Example 16. The resulting reaction mixture was dried toobtain a dry surfactant past which was re-dispersed in an oil of choice.An aqueous solution of monomer(s) and or preformed polymer(s), across-linking agent and initiator were added. The mixture was subjectedto a polymerization step, and the polymerized reaction product wasdried. The dried mass was dispersed in aqueous buffer; and the methodscan be combined with READ to implement multi-step on-chip assaymanipulations.

Example 16b: Coupling of a Biomolecules

This Example illustrates the coupling of a biomolecule to the magneticnanoparticles of the invention. The magnetic nanoparticle of Example 16acan be further functionalized to perform an assay for interest bybinding to the functional site on the magnetic nanoparticle certainmolecules such as for example DNA (oligonucleotides) or RNA fragments,peptides or proteins, aptamers and small organic molecules. The bindingof such molecules can be performed using processes known in the art, forexample, using one or several coupling reactions (See, e,g, G. T.Hermanson, Bioconjugate Techniques (Academic Press, 1996); L. Illum, P.D. E. Jones, Methods in Enzymology 112, 67-84 (1985)). Attachment of themolecule of interest to the functional site generally requires aone-step or two-step reaction which can be performed in parallel usingstandard liquid handling robotics and a 96-well format to covalentlyattach any of molecules to the functional site on the magneticnanoparticle.

Specifically, this Example illustrates a method of attaching a probe(for example a protein), to the functional site of a magneticnanoparticle of the invention, using well-established carbodiimidechemistry. In a 2 ml vial an aliquot containing 10 mg of carboxylatefunctionalized magnetic nanoparticle was mixed with 1 ml 10 mM boratebuffer (pH=8.5). The resulting particles were then separatedmagnetically using a permanent magnet separator and the supernatant wassiphoned off. The separated pellet was washed two times (using the sameprotocol as above) in 0.1M MES buffer (pH=4.5) and finally re-suspendedin 600 μl of the same. In a separate vial 3 mg of Neutravidin (a biotinbinding protein, Pierce Chemicals, Rockford, Ill.) was dissolved in 300μl of the MES buffer and the solution was slowly added to the suspensionof the magnetic charged particle. The suspension is sonicated using aprobe sonicator. Following sonication, EDAC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (Aldrich-Sigma,Milwaukee, Wis.) solution (200 mg/ml) was added. The mixture was allowedto react for 2 hours at room temperature, following which the resultingmagnetic nanoparticles were magnetically separated, washed one time witha coupling buffer, two times in a borate buffer and resuspended andstored in a storage buffer (PBS pH=7.4, 0.1% (w/v) BSA, 0.5% (w/v) Tween20, 10 mM EDTA and 0.02% (w/v) NaN₃) at 2-8° C.

Example 16c: Preparation of Composite Particles

This Example illustrates the production of encoded magnetic articles.Encoded particles were reacted with the magnetic nanoparticles ofExample 16b. In a more general embodiment, super-paramagnetic polymernanoparticles may also be used (such as those obtained from MACSmicrobeads, Miletnyi Biotech Inc, Auburn, Calif.; Captivate ferrofluidparticles, Molecular Probes, Eugene, Oreg.; Nanomag particles, Micromod,Rostock, Germany).

FIG. 23 illustrates a sub-library of 12 types of encoded magneticparticles. The encoded particles were synthesized using 3.2 μm diametercross-linked polystyrene particles with tosyl surface groups (BangsLabs, Fishers, Ind.) and two hydrophobic pyrromethane dyes which wereeach introduced by swelling and bulk staining. The dyes wereindividually introduced to obtain four intensity levels, and were mixedin four nominal molar ratios. Following, 100 μl of a 1% solution of thecolored Latex (in PBS) was mixed with 100 μl or 200 μl of Captivateferrofluid (streptavidin conjugate, 200 nm diameter, used as supplied)(Molecular Probes, Eugene, Oreg.) or 100 μl of Nanomag dextran coated.Streptavidin functionalized magnetite particles 130 nm diameter wereused as supplied) (Nanomag particles, Micromod, Rostock, Germany). Thewhole reaction volume is made up to 500 μl using phosphate buffer pH 7.8and the coupling reaction is allowed to proceed overnight. The resultingparticles are separated magnetically washed 1×, resuspended and storedinstorage buffer (PBS pH=7.4, 0.1% (w/v) BSA, 0.5% (w/v) Tween 20, 10 mMEDTA and 0.02% (w/v) NaN₃) at 2-8° C.

Example 16d: Controlling the Content of Metal Oxide

This Example illustrates the method by which the content of the metaloxide can be controlled to impart on the magnetic moment of the encodedmagnetic particle of the invention. This can be accomplished by varyingthe number of magnetic nanoparticles which can bind to the surface ofthe encoded particle For ease of monitoring, and by way of illustration,instead of magnetic nanoparticles, Fluoronanogold, a fluorescein-goldnanoparticle—streptavidin conjugate (supplied from Nanoprobes, Yaphank,N.Y.) was used to monitor the number of particles. The reagent contained˜0.08 mg/ml of streptavidin and was used as received.

In this Example, 0.5 mg of biotin functionalized 3.36 μm diameterpolystyrene beads (Spherotech, Libertyville, Ill.) was suspended in 500μl of 20 mM phosphate buffered saline (150 mM) at pH 7.4 with 0.1% BSA.Varying amounts of Fluoronanogold reagent were added to the beadsolution and the streptavidin-biotin binding reaction allowed to proceedfor 1 hour at room temperature, following which the microparticles wereseparated by centrifugation and supernatant removed. FIG. 24 shows thefluorescence intensity from the particles bound on the surface as afunction of the amount of Fluoronanogold reagent added.

Example 17: Synthesis of Fluorescently Colored and Magnetic Polymer BeadComposites

A stock solution of hydrophobic fluorescent dye and the iron oxideparticles was made by re-dispersing the dried magnetic composite and thedye in the solvent of choice, for example a CHCl₃ (Aldrich Chemical Co.,Milwaukee, Wis.) or CH₂Cl₂/CH₃OH mixture (70/30 (v/v)) (Aldrich ChemicalCo., Milwaukee, Wis.). A predetermined amount of polymer beads waswashed thoroughly in methanol (3×) and then evaporated dry. Simultaneousincorporation of the fluorescent dye and the iron oxide nanoparticle wasachieved by swelling the beads in organic solvent/nanoparticle/dyemixture. The swelling process was completed within ˜1 hr. Following thisthe polymer beads were separated by centrifugation and washed withmethanol (3×) followed by isooctane (2×) and then methanol (2×) andfinally redispersed in 0.2% SDS-DI water solution.

Example 18: Formation of Arrays by Application of a Magnetic Field

The composite particles of the invention when exposed to a homogeneousaxial magnetic field (oriented normal to the substrate plane), can bearranged in 2D array format. As a function of increasing magnetic fieldstrength which is dependent on the paramagnetic susceptibility of thecomposite particle, planar assemblies can be formed. Permanent magnetscan be designed so as to produce the field strength required to realizethe desired configuration of the assembly. Requisite magnetic fieldconfigurations can also be produced by an electromagnet in solenoid orHelmholtz configuration known to the art; the substrate can beintroduced into the magnet bore or can be placed in immediate proximityto the coil(s) outside of the bore so as to ensure the orientation ofthe field substantially normal to the substrate plane. Spatiallymodulated magnetic fields can be produced by patterning the substratewith perm-alloy using methods known to the art.

In this Example, three differently sized composite particles withwherein the encoded particles are 3.2 microns, 6.9 microns and 10.9microns were synthesized as described herein, as expected, themagnetization of the composite particles increased with increasingparticle size.

FIG. 25 shows a 2D geometry of 3.2 microns composite particle exposed toa magnetic field of approximately 1000 Gauss produced using a permanentmagnet.

Example 19: Two-Dimensional Colloidal Arrays: Formation of Magnetic“Wigner” Crystals

This Example illustrates the ability to manipulate the compositeparticles of the invention. Exemplification is provided by themanipulation of superparamagnetic particles suspension such assuperparamagnetic Oligo(dT)₂₅ microparticle (Dynal, Lake Success, N.Y.).Suspension of these particles are formed by mixing the particle at aconcentration of. 5×10⁷ beads/ml) in 0.01 mM saline, containing 0.05%triton-X100 (Sigma-Aldrich, Milwaukee, Wis.) as a stabilizer. Themagnetization of the particles is completely reversible and, at lowfield strengths, is proportional to the external field through theeffective volumetric magnetic susceptibilty (=0.192). The suspensionsare held on a microscope stage in a sandwich cell formed by a siliconelectrode and an ITO coated glass electrode separated by an adhesivespacer ˜100 μm thick. A nominally uniform magnetic field is generated inthe sample by a coil of copper-wire placed underneath the sandwich cell.After application of the magnetic field the evolution of the suspensionstructure is recorded with a CCD camera attached to a VCR and the imagesdigitized for further analysis. FIG. 26 shows the experimental setup andFIG. 27 shows a series of snapshots illustrating the formation of2-dimensional magnetic arrays.

When a magnetic field is applied, the particles acquire magnetic momentsm=4/3πa³μ_(o)χH, where a is the particle radius, μ_(o) the magneticpermeability of vacuum, and H is the external field.

Example 20: Streptavidin-Biotin Binding Assay Using Fluorescent MagneticMicroparticles

In this Example, to 100 μl of a suspension of the composite particles ofthe invention, which have been functionalized with neutravidin asdescribed in Example 16c (1%) solids is added to a 1.5 ml vial and thesuspension diluted with 900 μl of PBS with 0.01% (w/v) of Tween-20(PBST). The composite particles of the invention are mixed by vortexingand then separated magnetically and the supernatant aspirated off. Thepellet is resuspended in 980 μl of PBS. 20 μl of abiotin-Oligo(dT)₅-CY5.5 (IDT, Coralville, Iowa) at a concentration (26.7ng/ml). The mixture is incubated for 30 minutes at room temperature.Following this the particles of the invention are separated magneticallyand washed 2× in PBST and resuspended in 1 ml of PBST. The particles ofthe invention are then assembled on a chip and their surfacefluorescence estimated using the method previously described. Theresults are shown in FIG. 28. For purposes of comparison, the encodedparticles of the invention in the absence of the magnetic nanoparticlesof the invention, but having covalently attached thereon neutravidinwere also reacted to the biotinylated probe using the proceduredescribed in this Example.

Example 21: On-Chip Hybridization of Target Molecules

This Example relates to on-chip hybridization of target molecules tooligonucleotides probes immobilized to the composite particles of theinvention. Biotinylated oligonucleotides with known base sequence werefirst coupled to a given type of color-coded composite particles thathave been previously coated with Neutravidin on the surface. Thecoupling reaction was carried out in 0.1 ml coupling buffer (150 mMNaCl, 0.05 mM ethyenediamine tetra-acetic acid (EDTA), 0.5% bovine serumalbumin, 0.5 mM Tris-HCl, and 100 mM sodium phosphate, pH 7.2) with 0.4μM biotinylated oligonucleotides, and approximately 6.7×10⁵ particles.The coupling reaction mix was incubated at room temperature for 30 minwith vortexing. Upon completion of the coupling reaction, the particleswere collected by centrifugation. The unreacted sites of theNeutriAvidin on the surface were blocked by using 0.1% biotin in 150 mMNaCl and 100 mM sodium phosphate, pH 7.2 with 0.05% Tween-20. Theblocking was carried out at room temperature for 20 min with vortexing.After blocking, the particles were washed with 0.2 ml of 150 mM NaCl and100 mM sodium phosphate, pH 7.2 with 0.05% Tween-20. The foregoingprocedure can be utilized to couple any biotinylated oligonucleotides ofinterest to different types of Neutravidin-coated particles of theinvention.

Several types of color-encoded particles of the invention were coupledwith biotinylated oligonucleotides with known sequences and werecombined into one tube for assembly of arrays on silicon chips. Thearrays thus formed were then used for on-chip hybridization of peptidenucleic acid (PNA) oligomers to specific complimentary oligonucleotidespreviously coupled to the micro particles of the invention.Specifically, hybridization was carried out in 30 μl hybridizationsolution (90 mM NaCl, 83 mM guanidine thiocyanate, 8 mM MgCl₂, 17 nMEDTA, 0.02% biotin, 0.1% Tween-20, 70 mM mM Tris-HCl, pH 7.5,) with 218nM biotinylated PNA oligomers. The arrays in the hybridization mix wereincubated at 40° C. for 60 min. Upon completion of hybridization, thearrays were washed with 50 μl of 250 mM NaCl, 10 mM Tris-HCl, pH 7.5,0.1% Tween-20, at room temperature for 10 min. For detection of thebiotinylated PNA oligomers that were hybridized on the compositeparticles of the invention, the arrays were incubated withCy5.5-conjugated Streptavidin (18 μg/ml) in 150 mM NaCl and 100 mMsodium phosphate, pH 7.2, at room temperature for 30 min. After washingwith 15 mM NaCl, 10 mM Tris-HCl, pH7.5, the arrays were examined byusing a fluorescence microscope. Fluorescence emitted from the particlesof the invention and the Cy5.5-labeled PNA oligomers were determined byusing optical filters with specific wavelengths. The particles of theinvention were decoded according to their color codes, and Cy5.5fluorescence emitted from specific particles was evaluated by using acomputer program (READ). Color-encoded particles devoid of magneticcharacter were used as controls in the assay. Results of the assay areshown in FIG. 29 demonstrate that PNAs specifically hybridize tocomplimentary oligonucleotides coupled on the composite particles.Fluorescence signal intensity was determined from four types of theparticles of the invention (I, II, III and IV) from two arrays (Chip Aand B, for panels A and B, respectively) in the on-chip hybridizationassay. The type I particles are the composite particles of theinvention, whereas Types II, III and IV are three different types of theencoded particles of the invention. Types I and II are functionalizedencoded particles with biotinylated oligonucleotides that arecomplementary to the PNA oligomers. Type III encoded particles arefunctionalized with an oligonucleotide with unrelated base sequence tothe PNA. Type IV encoded particles have no oligonucleotides on theirsurface. Chip B serves as a negative control for chip A, which wasincubated with the hybridization mixture but without the target PNA. Thesymbol “n” denotes the number of particles on each type and the barsrepresent standard deviation of the mean.

Example 22: Immunoassay Using Fluorescent Magnetic Microparticles

In this Example, the composite particles of the invention are used forcarrying-out immunoassays. The composite particles of the invention canbe customized to display antibody of interest on their surface. An arrayof composite particles of the invention can be exposed to the samplesolution (such as serum solution) containing antigens of interest.Subsequent addition of fluorescently labeled secondary antibodiesresults in the formation of ternary fluorescent complex theconcentration of which can be monitored by recording the fluorescencefrom the composite particle surface using methods disclosed in thisinvention. Arrays of the antibody functionalized composite particles ofthe invention can thus be used for monitoring serum protein levels, andthe binding pattern of the antigens of interest may be useful in proteinprofiling.

Example 23: Alternative I: Magnetic Field-Induced Array Formation

This Example illustrates the operation of FIG. 22 and a sequence ofreaction steps leading from the sequence-specific capture of mRNAmolecules to the formation of a EFM bead-displayed cDNA array. Such anarray will be useful in many applications. For example, in geneprofiling, a planar cDNA array will be useful to profile mRNAconcentrations; alternatively, the cDNA array can be probed directlyusing a pool of labeled DNA binding agents or probes to profile thegenes of interest within the array. The protocol, performed in atemperature controlled device, comprises the following steps: (1)introducing a set of the composite particle of the invention into afirst compartment containing a pool of mRNA molecules, each particletype displaying a gene-specific oligonucleotide probe; mRNA moleculesare permitted to anneal to their corresponding probes. Assay conditionsfor this capture step are known in the and are applicable herein; (2)perform on-particle reverse transcription (RT) using particle-attachedmRNA as template and release mRNA from the particles; (3) applyingmagnetic field from permanent magnet and wash while retaining theparticles; (4) releasing the magnetic field and re-suspending theparticles in buffer to perform the process of LEAPS; (5) direct theparticles to a compartment containing custom-designed chips and form aplanar array of the composite particles of the invention using LEAPS.

Example 24: Alternative II: Magnetic Field-Induced Array Formation

This Example illustrates the operation of FIG. 22 and a sequence ofreaction steps leading from the sequence-specific capture of mRNAmolecules to the formation of a EFM particle-displayed cDNA array. Theprotocol set forth in Example 21 is used except that a planar array ofthe composite particles of the invention by applying a magnetic field tois formed using the application of a magnetic field and steps (4) and(5) of Example 21 are omitted.

Example 25: Multistep Assay Sequence Using Arrays of Encoded MagneticBeads

This Example illustrates a multi-step biochemical reaction protocolwhich integrates the capture of genomic DNA fragments to a first set ofmagnetic nanoparticles, followed by magnetic separation and solid-phaseamplification of captured “particle-tagged” fragments according to thegeneral process described in FIG. 22. This capture step is followed by atransformation step of concurrent “multiplex” PCR amplificationreactions, each reaction containing a small set of primer pairs toproduce solution-borne amplicons. These are pooled and placed intocontact with a set of the composite particles of the invention formultiplexed analysis invoking post-transformation mediated byapplication of a magnetic field.

The foregoing can be accomplished by the following steps:

1. Sample Capture and First Transformation

Given a solution containing fragments of genomic DNA, divide into fourequal parts, aliquots are injected into four separate reactioncompartments, equipped to permit temperature and control in accordancewith standard PCR temperature cycling protocols. Into each compartment,one or more magnetic nanoparticles—tagged primers are injected. Aparticle-tagged primer is composed of magnetic nanoparticles displayingan oligonucleotide probe directed against a genomic DNA target fragmentof interest, said probe also serving as a first primer of a subsequentpolymerase-catalyzed primer extension reaction. The next step permitscapture by hybridization of selected fragments to matchingparticle-displayed probes, said targets serving as templates insubsequent extension reaction. To each compartment, polymerase is addedand one or more second primers as needed to permit template-directedextension of particle-coupled capture probes under temperature cyclingconcurrently in all compartments, thereby producing first extensionproducts, anchored to the magnetic nanoparticles. A magnetic field isapplied to form a planar array of magnetic nanoparticles in eachcompartment. The compartments are washed while retainingparticle—anchored extension products

A typical PCR protocol using particle-tagged primers to amplify afragment of genomic DNA is as follows. An oligonucleotide probecorresponding to a specific target position within the fragment ofinterest is designed with variable 3′ terminus aligned at or near thetarget position. Probes are synthesized to contain a 5′ biotin-TEG witha 12 C spacer (Synthegen Tex.). Probes are attached tostreptavidin-coated magnetic beads in accordance with standard reactionprotocols such as the following: add to a magnetic bead suspension in1×TE (100 mM Tris-HCl, 10 mM EDTA), 500 mM NaCl for 45 minutes at roomtemperature; wash beads with 1×TE, 150 mM of NaCl for 3×; and suspend in50 μl of same solution. Next, add 1 μl of each bead suspension to to PCRmix containing 1× buffer (100 mM Tris-HCl, pH. 9.0, 1.5 mM MagnesiumChloride, 500 mM KCL), 40 μM Cy5-labeled dCTP (Amersham PharmaciaBiotech N.J.), and 80 μM of the remaining three dNTPs, and 3 units ofTaq DNA polymerase (Amersham Pharmacia Biotech N.J.). Genomic DNA targetfragments (40 ng) are added to the PCR mix just prior to initiatingextension. Under these conditions, ten cycles of amplification sufficeto produce sufficient bead-anchored extension product using a PerkinElmer 9600 thermal cycler, a temperature cycle consisting ofdenaturation (30 s at 90 C), annealing (30 s at 55 C), and extension (20s at 72 C). Following completion of the extension reaction, beadstypically are washed four times by centrifugation in 1×TE buffer.

2. Concurrent Instances of Second Amplification

To each reaction compartment, polymerase was injected along with a setof one or more primer pairs designed to select regions withinparticle-anchored first extension products, each compartment receiving aunique set of such primer pairs. concurrent amplification ofbead-anchored fragments under temperature cycling is allowed to occur,thereby producing, in each compartment, a set of specific amplicons asdirected by the corresponding primer sets admitted into the compartment.Standard PCR reaction protocols (REF) apply to this step.

3. Pooling of Reaction Products, Post-Assay Array Assembly and Detection

Solutions containing amplicons are combined to produce analyte solutionand transfer to a detection compartment and placed into contact with aset of the encoded and magnetic nanoparticles of the invention, eachparticle displaying a sequence-specific oligonucleotide probe uniquelydirected against one of the amplicons within the analyte solution.Annealing of amplicons to bead-displayed probes to form hybridizationcomplexes is permitted to occur, and analysis of selected regions oftarget sequence by method of choice, e.g., differential thermalstability of hybridization complexes or probe elongation is performed. Amagnetic field is applied to form planar array of the particles of theinvention. An assay image signal is recorded. A decoding image isrecorded to permit decoding of capture probe identities

Standard assay conditions for polymorphismrhism analysis apply to thisstep, providing also conditions permitting formation of planar arrays ofencoded functionalized magnetic beads, such conditions being illustratedherein in connection with Example NN and Figure MM (SB: EFM bead array,induced by permanent magnet.)

Example 26: Mixed Clusters and Arrays

This Example illustrates an assay format wherein target DNA strands areattached to magnetic nanoparticles of the invention. Chemical attachmentis readily accomplished using standard protocols of bioconjugatechemistry as known in the art. For example, strands of DNA are readilybiotinylated and may then be attached to streptavidin-coated magneticnanoparticles. Alternatively, particle-displayed DNA strands areproduced by PCR using bead-tagged primers, as discussed in Example 21.The requisite set of encoded particles of the invention are produced bythe methods provided herein. The particles are functionalized withspecific oligonucleotide probes using standard methods of bio-conjugatechemistry as know in the art. In the course of the assay,hetero-structures composed of magnetic nanoparticles and encodedparticles are formed as a result of the formation of a complex betweenbinding partners, and these complexes are assembled into a planar arrayfor detection.

Two alternatives are set forth herein. In the first alternative, thesize of the optically encoded beads will typically significantly exceedthat of the magnetic beads, a typical ratio of respective radii being100:1; streptavidin-coated magnetic nanoparticles of 100 Å diameter arecommercially available, as herein discussed in connection with Example22. Next, a mixture of magnetic bead-displayed DNA target strands iscombined with a set of color-encoded beads displaying oligonucleotideprobes under conditions permitting hybridization of matching magneticbead-tagged target targets to probes. Formation of hybridizationcomplexes will “decorate” each color-encoded bead with a large number ofmagnetic nanoparticles to produce a magnetic shell, resulting in theformation of a particle of the structure and composition discussedherein.

Application of a magnetic field in accordance with the methods of thepresent invention, will then produce a planar array of the encoded andmagnetic nanoparticles of the invention to permit recording of amulticolor fluorescence image to identify captured DNA targets.Alternatively, assay conditions may be chosen so as to favor theformation of clusters. That is, the magnetic nanoparticles of theinvention acting as multi-dentate “ligands”, mediate “agglutination” ofthe encoded particles of the invention into a cluster of the encoded andmagnetic of particles of the invention. Application of a strong magneticfield, typically producing magnetization in excess of 1000 Gauss,permits separation of these clusters from solution into a an expandedassembly of isolated clusters.

In a second alternative, the roles, and hence the relative sizes, ofmagnetic and encoded particles of the invention are reversed so thatlarger magnetic nanoparticles are decorated by the encoded particlesApplication of a magnetic field in accordance with the conditions setforth in Example 19, (Dynal beads—solenoid) herein produces a planararray of magnetic nanoparticles wherein each particle will display anoptical signal or not display an optical signal depending on whether theparticle have been endowed with an optical identifier.

Both alternatives in this Example require reading of multi-opticalsignatures to identify captured targets. When particle optical loadingencoding is achieved by application of a small number of encodingcolors, typically two or three, and variation of the ratio of suchencoding colors in accordance with standard methods as known in the art,multicolor imaging is readily achieved by standard multicolorfluorescence imaging using multiple filter sets as known in the art.Otherwise, if combinations of multiple distinct colors are used toproduce the color code, then methods of multi-spectral imaging aregenerally available to record the assay image.

We claim:
 1. A method of multiplex analysis of analytes in a solution,comprising: providing a plurality of magnetically polarizablemicroparticles of two or more types wherein different types bear anoptically distinguishable signature, and the different types displaydifferent capture moieties on their surfaces capable of binding todifferent analytes; suspending the microparticles in a first solutioncontaining, or suspected to contain, analytes of interest, underconditions permitting the capture of analytes by the capture moieties,and wherein an optical signal is generated following such capture; usinga magnetic field to assemble the microparticles in a planar array on adesignated section of a substrate, where said magnetic field isgenerated by coils or magnets, and is uniformly distributed over thesurface of the substrate, and wherein the spacing between particleswithin the array is varied by varying the strength of the magneticfield; and imaging the optically distinguishable signatures associatedwith the microparticles and the optical signals, and correlating theoptical signals with microparticles having particular opticallydistinguishable signatures to determine which analytes are bound bywhich capture moieties.
 2. The method of claim 1 wherein the opticalsignals arise as a result of the binding of an analyte by a capturemoiety.
 3. The method of claim 2 wherein the optical signal indicatesthe transformation of the capture moiety mediated by the binding of theanalyte.
 4. The method of claim 1 wherein the first solution is removedand replaced with a second solution prior to imaging the opticallydistinguishable signatures associated with the microparticles and theoptical signals.
 5. The method of claim 1 wherein array assembly isinitiated at a preselected time by actuating a magnetic field.